Yeast-brachyury immunotherapeutic compositions

ABSTRACT

Disclosed are yeast-based immunotherapeutic compositions comprising Brachyury antigens, and methods for the prevention and/or treatment of cancers characterized by the expression or overexpression of Brachyury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/803,719, filed Mar. 14, 2013, now U.S. Pat. No. 9,198,719 whichclaims the benefit of priority under 35 U.S.C. §120 and is acontinuation of PCT Application No. PCT/US12/29636, filed Mar. 19, 2012,which claims the benefit of priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/453,656, filed Mar. 17, 2011. Theentire disclosure of each of U.S. application Ser. No. 13/803,719, PCTApplication No. PCT/US12/29636 and U.S. Provisional Application Ser. No.61/453,656 is incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was created in the performance of a Cooperative Researchand Development Agreement with the National Institutes of Health, anAgency of the Department of Health and Human Services. The Government ofthe United States has certain rights in this invention.

STATEMENT REGARDING JOINT RESEARCH AGREEMENT

This invention was made by or on behalf of parties to a CooperativeResearch and Development Agreement, executed May 8, 2008. The parties tothe Cooperative Research and Development Agreement are: GlobeImmune,Inc. and the U.S. Department of Health and Human Services, asrepresented by National Cancer Institute, an Institute, Center orDivision of the National Institutes of Health.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “3923-34-PCT_ST25”, has asize in bytes of 76 KB, and was recorded on 13 Mar. 2012. Theinformation contained in the text file is incorporated herein byreference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to yeast-based immunotherapeuticcompositions and methods for the prevention and/or treatment of cancerscharacterized by the expression or overexpression of Brachyury.

BACKGROUND OF THE INVENTION

Brachyury, also known as “T”, is a mesodermal transcription factor andmember of the T-box complex of genes. The gene encoding Brachyury(denoted as either T gene or Brachyury gene in humans) was initiallyidentified in 1927 by Nadine Dobrovolskaïa-Zavadskaïa through a mutationin mice that affected tail length and sacral vertebrae in heterozygousanimals. The Brachyury gene was cloned in mice in 1990 by Hermann andcolleagues (Herrmann et al., 1990, Nature 343:617-622) and in humans in1996 by Edwards and colleagues (Edwards et al., 1996, Genome Res.6:226-223), who also described the deduced amino acid sequence for humanBrachyury.

As a member of the T-box family of transcription factors, Brachyurycontains the highly conserved DNA-binding domain motif, called “T-box”or T-domain, which binds to a palindromic consensus sequence. Brachyury,like other T-box proteins, has been shown to play a role in earlydevelopment, and is vital for the formation and differentiation ofposterior mesoderm and axial development in vertebrates (see, e.g.,Wilkinson et al., 1990, Nature 343(6259):657-659); Beddington et al.,1992, Development (Suppl.):157-165; Schulte-Merker et al., 1994,Development 120: 1009-1015; Kispert and Herrmann, 1994, Dev. Biol.161:179-193; Showell et al., 2004, Dev Dyn 229:201-218). More recently,Palena and colleagues have demonstrated that Brachyury is expressed in avariety of human tumor tissues and cancer cell lines and have shown thatpeptides of Brachyury can be used to generate Brachyury-specific T celllines in normal donors and cancer patients (Palena et al., 2007, Clin.Cancer Res. 13(8):2471-2478). Studies by Fernando et al. have shown thatBrachyury promotes the epithelial-mesenchymal transition (EMT) in humantumor cells, conferring on tumor cells a mesenchymal phenotype, as wellas migratory and invasive abilities, while attenuating tumor cell cycleprogression (Fernando et al., 2010, J. Clin. Invest. 120(2):533-544).Accordingly, Brachyury is involved in metastatic progression of cancer.

Cancer is a leading cause of death worldwide, and the development ofeffective therapies for cancer continues to be one of the most activeareas of research and clinical development. Although a variety ofinnovative approaches to treat and prevent cancers have been proposed,many cancers continue to have a high rate of mortality and may bedifficult to treat or relatively unresponsive to conventional therapies.Cancers associated with Brachyury expression may be found in a varietyof tissues, including breast, small intestine, stomach, kidney, bladder,uterus, ovary, testes, lung, colon and prostate, and includes metastaticand late-stage cancers. In addition, Brachyury is expressed in tumors ofB cell origin, such as chronic lymphocytic leukemia (CLL), Epstein-Barrvirus transformed B cells, Burkitt's and Hodgkin's lymphomas. Therefore,Brachyury appears to play a role in a large number of human cancers.While Brachyury has been proposed to be a target for cancerimmunotherapy (see, e.g., Palena et al., supra, Fernando et al., supra,and WO 2008/106551), since this is a relatively new cancer target, thereremains a need in the art for new immunotherapeutic products thateffectively treat and/or prevent cancers associated with Brachyuryexpression or overexpression.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a method to reduce, arrest,reverse, delay or prevent the metastatic progression of cancer in anindividual who has cancer. The method includes the step of administeringto an individual who has a cancer that is undergoing metastaticprogression, is at risk of undergoing metastatic progression, or ispredicted to begin undergoing metastatic progression, animmunotherapeutic composition comprising: (a) a yeast vehicle; and (b) acancer antigen comprising at least one Brachyury antigen. Anotherembodiment of the invention relates to the use of an immunotherapeuticcomposition comprising a yeast vehicle and a cancer antigen comprisingat least one Brachyury antigen to reduce, arrest, reverse or prevent themetastatic progression of cancer in an individual who has cancer.

In one aspect, of these embodiments of the invention, Brachyury is notdetected in the individual's cancer at the time the composition is firstadministered. In one aspect, Brachyury expression is detected in theindividual's cancer at the time the composition is first administered.The individual may have stage I cancer, stage II cancer, stage IIIcancer, or stage IV cancer.

Another embodiment of the invention relates to a method to prevent ordelay the onset of a Brachyury-expressing cancer. The method includesthe step of administering to an individual an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a cancer antigencomprising at least one Brachyury antigen. Another embodiment of theinvention relates to the use of an immunotherapeutic compositioncomprising a yeast vehicle and a cancer antigen comprising at least oneBrachyury antigen to prevent or delay the onset of aBrachyury-expressing cancer.

In one aspect of these embodiments, cancer has not been detected in theindividual. In one aspect, the individual is at high risk for developingcancer (e.g., via a genetic predisposition). In one aspect, theindividual has a pre-cancerous lesion.

In one aspect of these embodiments, the individual has cancer, butBrachyury-expressing cancer cells have not been detected in the cancer.In one aspect, the cancer is not yet metastatic. In one aspect, thecancer has a high risk of metastasizing. In one aspect, the subject hasstage I cancer. In one aspect, the subject has stage II cancer.

Another embodiment of the invention relates to a method to reduce orprevent chemotherapy-resistance or radiation-resistance of tumor cellsin a patient with cancer. The method includes the steps of administeringto an individual who has cancer and is receiving chemotherapy and/orradiation therapy an immunotherapeutic composition comprising: (a) ayeast vehicle; and (b) a cancer antigen comprising at least oneBrachyury antigen. Another embodiment of the invention relates to theuse of an immunotherapeutic composition comprising a yeast vehicle and acancer antigen comprising at least one Brachyury antigen to reduce orprevent chemotherapy-resistance or radiation-resistance of tumor cellsin a patient with cancer. In one aspect of this embodiment of theinvention, Brachyury is not detected in the individual's cancer at thetime the composition is first administered. In one aspect, Brachyuryexpression is detected in the individual's cancer at the time thecomposition is first administered.

Yet another embodiment of the invention relates to a method to treatcancer. The method includes the steps of: (a) administering to anindividual who has cancer in which Brachyury expression has not beendetected, a first immunotherapeutic composition comprising a yeastvehicle and a first cancer antigen that does not comprise a Brachyuryantigen; and (b) administering to the individual, prior to, concurrentlywith, sequentially with, or subsequent to, administration of the firstimmunotherapeutic composition a second immunotherapeutic compositioncomprising a yeast vehicle and a second cancer antigen comprising aBrachyury antigen. In one aspect, the method further comprises, in step(a), administering one or more additional immunotherapeuticcompositions, wherein the each of the one or more additionalimmunotherapeutic compositions comprises an additional cancer antigen.In one aspect of either embodiment above, the cancer antigen is selectedfrom: mutated Ras, carcinoembryonic antigen (CEA), MUC-1, EGFR, BCR-Abl,MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, PSMA, tyrosinase, TRP-1(gp75), NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2,hTERT, p73, B-RAF, adenomatous polyposis coli (APC), Myc, vonHippel-Lindau protein (VHL), Rb-1, Rb-2, androgen receptor (AR), Smad4,MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H, HERV-K,TWIST, Mesothelin, and NGEP. In one aspect, the cancer antigen isselected from the group consisting of: mutated Ras, carcinoembryonicantigen (CEA), and MUC-1. Another embodiment of the invention relates tothe use of a combination of immunotherapeutic compositions to treatcancer, the immunotherapeutic compositions comprising: (a) a firstimmunotherapeutic composition comprising a yeast vehicle and a firstcancer antigen that does not comprise a Brachyury antigen; and (b) asecond immunotherapeutic composition comprising a yeast vehicle and asecond cancer antigen comprising a Brachyury antigen.

Yet another embodiment of the invention relates to a method treatcancer. The method includes the steps of: (a) administering to anindividual who has cancer a first immunotherapeutic compositioncomprising a yeast vehicle and a mutated Ras antigen; (b) administeringto the individual of (a) a second immunotherapeutic compositioncomprising a yeast vehicle and an antigen selected from the groupconsisting of carcinoembryonic antigen (CEA) and mucin-1 (MUC-1); and(c) administering to the individual of (a) and (b) a thirdimmunotherapeutic composition comprising a yeast vehicle and a Brachyuryantigen. In one aspect, the steps of administration in (a), (b) and (c)are concurrent. Another embodiment of the invention relates to the useof a combination of immunotherapeutic compositions to treat cancer, theimmunotherapeutic compositions comprising: (a) a first immunotherapeuticcomposition comprising a yeast vehicle and a mutated Ras antigen; (b) asecond immunotherapeutic composition comprising a yeast vehicle and anantigen selected from the group consisting of carcinoembryonic antigen(CEA) and mucin-1 (MUC-1); and (c) a third immunotherapeutic compositioncomprising a yeast vehicle and a Brachyury antigen.

In any of the embodiments or aspects of the invention described above orelsewhere herein, where the individual has cancer or a precancerouslesion, in one aspect of the invention, the individual is being treatedor has been treated with another therapy for cancer. For example, such atherapy can include, but is not limited to, chemotherapy, targetedcancer therapy, radiation therapy, adoptive T cell transfer, and/oradministration of one or more additional immunotherapeutic compositions.In one aspect, an additional immunotherapeutic composition comprises ayeast vehicle and a second cancer antigen that does not includeBrachyury antigen. The second cancer antigen can include, but is notlimited to, mutated Ras, carcinoembryonic antigen (CEA), MUC-1, EGFR,BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, PSMA, tyrosinase,TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA,HER-2/neu/c-erb/B2, hTERT, p73, B-RAF, adenomatous polyposis coli (APC),Myc, von Hippel-Lindau protein (VHL), Rb-1, Rb-2, androgen receptor(AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H,HERV-K, TWIST, Mesothelin, and NGEP. In one aspect, the second cancerantigen is selected from: mutated Ras, carcinoembryonic antigen (CEA),and MUC-1.

In one aspect of any of the embodiments or aspects of the inventiondescribed above or elsewhere herein, the method or use reduces tumorburden in the individual, increases survival of the individual, and/orinhibits tumor growth in the individual.

In one aspect of any of the embodiments or aspects of the inventiondescribed above or elsewhere herein, the method further comprisessurgical resection of a tumor from the individual.

In one aspect of any of the embodiments or aspects of the inventiondescribed above or elsewhere herein, the cancer is of epithelial cellorigin. In one aspect, the cancer can include, but is not limited to,breast cancer, small intestine cancer, stomach cancer, pancreaticcancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer,testicular cancer, lung cancer, colon cancer, prostate cancer, chroniclymphocytic leukemia (CLL), Epstein-Barr virus transformed B cells,Burkitt's lymphoma, Hodgkin's lymphoma, or metastatic cancers thereof.

In one aspect of any of the embodiments or aspects of the inventiondescribed above or elsewhere herein, the Brachyury antigen isfull-length human Brachyury. In one aspect, the Brachyury antigen is notfull-length Brachyury. In one aspect, the Brachyury antigen has an aminoacid sequence represented by SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:2, oran amino acid sequence that is at least 95% identical to SEQ ID NO:6,SEQ ID NO:18, or SEQ ID NO:2. In one aspect, the Brachyury antigencomprises from at least position 1 or 2 to between position 255 and theC-terminus of SEQ ID NO:6, SEQ ID NO:18, or SEQ ID NO:2. In one aspect,the Brachyury antigen comprises from at least position 1 or 2 to betweenposition 430 and the C-terminus of SEQ ID NO:6, SEQ ID NO:18, or SEQ IDNO:2. In one aspect, the Brachyury antigen comprises positions 246 to254 of SEQ ID NO:6, SEQ ID NO:18, or SEQ ID NO:2. In one aspect, theBrachyury antigen comprises SEQ ID NO:6, positions 2-435 of SEQ ID NO:6,or an amino acid sequence that is at least 95% identical to SEQ ID NO:6.In one aspect, the Brachyury antigen comprises SEQ ID NO:18, positions2-435 of SEQ ID NO:18, or an amino acid sequence that is at least 95%identical to SEQ ID NO:18. In one aspect, the Brachyury antigencomprises SEQ ID NO:2, positions 2-435 of SEQ ID NO:2, or an amino acidsequence that is at least 95% identical to SEQ ID NO:2. In one aspect,the Brachyury antigen comprises SEQ ID NO:6, positions 2-435 of SEQ IDNO:6, or an amino acid sequence that is at least 99% identical to SEQ IDNO:6. In one aspect, the Brachyury antigen comprises SEQ ID NO:18,positions 2-435 of SEQ ID NO:18, or an amino acid sequence that is atleast 99% identical to SEQ ID NO:18. In one aspect, the Brachyuryantigen comprises SEQ ID NO:2, positions 2-435 of SEQ ID NO:2, or anamino acid sequence that is at least 99% identical to SEQ ID NO:2. Inone aspect, the cancer antigen is at least 25 amino acids in length. Inone aspect, the Brachyury antigen is at least 25 amino acids in length.In one aspect, the Brachyury antigen is greater than 30 amino acids inlength. In one aspect, the cancer antigen comprises two or moreimmunogenic domains of Brachyury.

In one aspect of any of the embodiments or aspects of the inventiondescribed above or elsewhere herein, the cancer antigen is a fusionprotein. In one aspect, the fusion protein has an amino acid sequencerepresented by SEQ ID NO:8, or an amino acid sequence that is at least95% identical to SEQ ID NO:8. In one aspect, the fusion protein has anamino acid sequence represented by SEQ ID NO:20, or an amino acidsequence that is at least 95% identical to SEQ ID NO:20.

Another embodiment of the invention relates to a yeast-Brachyuryimmunotherapeutic composition, wherein the immunotherapeutic compositioncomprises: (a) a yeast vehicle; and (b) an antigen expressed by theyeast vehicle and comprising at least one Brachyury antigen, wherein theBrachyury antigen comprises greater than 30 amino acids of an amino acidsequence represented by SEQ ID NO:6, SEQ ID NO:18 or SEQ ID NO:2. In oneaspect, the Brachyury antigen comprises an amino acid sequence that isat least 95% identical to SEQ ID NO:6, SEQ ID NO:18 or SEQ ID NO:2. Inone aspect, the Brachyury antigen comprises from at least position 1 or2 to between position 255 and the C-terminus of SEQ ID NO:6, SEQ IDNO:18 or SEQ ID NO:2. In one aspect, the Brachyury antigen comprisesfrom at least position 1 or 2 to between position 430 and the C-terminusof SEQ ID NO:6, SEQ ID NO:18 or SEQ ID NO:2. In one aspect, theBrachyury antigen comprises positions 246 to 254 of SEQ ID NO:6, SEQ IDNO:18 or SEQ ID NO:2. In one aspect, the Brachyury antigen comprises SEQID NO:6, positions 2-435 of SEQ ID NO:6, or an amino acid sequence thatis at least 95% identical to SEQ ID NO:6. In one aspect, the Brachyuryantigen comprises SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or anamino acid sequence that is at least 95% identical to SEQ ID NO:18. Inone aspect, the Brachyury antigen comprises SEQ ID NO:2, positions 2-435of SEQ ID NO:2, or an amino acid sequence that is at least 95% identicalto SEQ ID NO:2. In one aspect, the Brachyury antigen comprises SEQ IDNO:6, positions 2-435 of SEQ ID NO:6, or an amino acid sequence that isat least 99% identical to SEQ ID NO:6. In one aspect, the Brachyuryantigen comprises SEQ ID NO:18, positions 2-435 of SEQ ID NO:18, or anamino acid sequence that is at least 99% identical to SEQ ID NO:18. Inone aspect, the Brachyury antigen comprises SEQ ID NO:2, positions 2-435of SEQ ID NO:2, or an amino acid sequence that is at least 99% identicalto SEQ ID NO:2. In one aspect, the cancer antigen is a fusion protein.In one aspect, the fusion protein has an amino acid sequence that is SEQID NO:8 or an amino acid sequence that is at least 95% identical to SEQID NO:8. In one aspect, the fusion protein has an amino acid sequence ofSEQ ID NO:20 or an amino acid sequence that is at least 95% identical toSEQ ID NO:20. In one aspect, the yeast vehicle is a whole yeast. In oneaspect, the whole yeast is heat-inactivated.

Yet another embodiment of the invention relates to a yeast-Brachyuryimmunotherapeutic composition comprising: (a) a whole, inactivatedyeast; and (b) a Brachyury fusion protein comprising the amino acidsequence of positions 2-435 of SEQ ID NO:6. The expression of theBrachyury fusion protein is under the control of the promoter CUP1, theBrachyury fusion protein is expressed by the yeast, and the compositionelicits a Brachyury-specific T cell response. In one aspect, the fusionprotein comprises the amino acid sequence of SEQ ID NO:8.

Yet another embodiment of the invention relates to a yeast-Brachyuryimmunotherapeutic composition comprising: (a) a whole, inactivatedyeast; and (b) a Brachyury fusion protein comprising the amino acidsequence of positions 2-435 of SEQ ID NO:18. The expression of theBrachyury fusion protein is under the control of the promoter CUP1, theBrachyury fusion protein is expressed by the yeast, and the compositionelicits a Brachyury-specific T cell response. In one aspect, the fusionprotein comprises the amino acid sequence of SEQ ID NO:20.

In one aspect of any of the embodiments or aspects of the inventiondescribed above or elsewhere herein, the yeast vehicle is a whole yeast.In one aspect, the whole yeast is killed. In one aspect, the whole yeastis heat-inactivated. In one aspect, the yeast expresses the antigen. Inone aspect, the yeast is from a genus selected from the group consistingof: Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces,Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. In one aspect,the yeast is from Saccharomyces. In one aspect, the yeast is fromSaccharomyces cerevisiae.

In one aspect of any of the embodiments of the invention described aboveor elsewhere herein, the composition is formulated in a pharmaceuticallyacceptable excipient suitable for administration to a subject.

Yet another embodiment of the invention relates to the use of any of theyeast-Brachyury immunotherapeutic compositions described herein to treata disease. In one aspect, the disease is cancer. In one aspect, thedisease is associated with an infectious agent. In one aspect, thedisease is associated with a virus or viral infection. Such a virus caninclude, but is not limited to, Epstein Barr Virus (EBV).

Another embodiment of the invention relates to a method to treat orprevent a disease or condition associated with Epstein Barr Virus (EBV)infection. The method includes the step of administering to anindividual any of the yeast-Brachyury immunotherapeutic compositionsdescribed herein.

Yet another embodiment of the invention relates to a method to produce ayeast-Brachyury immunotherapeutic composition. The method includes thesteps of: (a) culturing yeast that have been transformed with arecombinant nucleic acid molecule encoding a Brachyury antigen under thecontrol of a CUP1 promoter in a suitable medium in the absence of CuSO₄until the yeast reach mid-log growth phase; (b) inducing expression ofthe Brachyury antigen in the yeast by adding CuSO₄ to the medium; (c)culturing the yeast after step (b) for up to between 6 and 8 hours; and(d) harvesting the yeast. In one aspect, the yeast in step (a) arecultured to a cell density of between 1.0 and 2.0 Y.U. per millilitertotal culture volume. In one aspect, the yeast in step (a) are culturedto a cell density of between 1.0 and 1.5 Y.U. per milliliter totalculture volume. In one aspect, the yeast are cultured in steps (a)-(c)in a medium where the pH is maintained at pH 5.5 or higher. In oneaspect, the method additionally includes a step of heat-inactivating theyeast after step (d). For example, in one aspect, the yeast areheat-inactivated at about 56° C. for about 1 hour. In a further aspectof this embodiment, the yeast can be formulated for injection with apharmaceutically acceptable excipient. In one aspect, the yeast are fromSaccharomyces. In one aspect, the yeast are from Saccharomycescerevisiae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a digitized image of a Western blot showing detection byanti-Brachyury of expression of Brachyury in a yeast-Brachyuryimmunotherapeutic composition, with both U2 and UL2 media.

FIG. 1B is a digitized image of a Western blot showing detection byanti-His of expression of Brachyury in a yeast-Brachyuryimmunotherapeutic composition, with both U2 and UL2 media.

FIG. 1C is a digitized image of a Western blot showing the robustexpression of Brachyury agonist antigen in GI-6305 using anti-His toidentify the hexahistidine tag on the Brachyury fusion protein.

FIG. 2 is a digitized image of a Western blot showing expression ofBrachyury in a yeast-Brachyury immunotherapeutic composition where thecell density at antigen induction and the time to harvest after antigeninduction were varied.

FIGS. 3A-3C are graphs showing that peripheral blood mononuclear cells(PBMCs) from two out of three healthy donors pulsed with yeast-Brachyuryfor two cycles of stimulation, followed by pulsing with Brachyury CTLpeptide, were capable of generating CD8⁺ CTLs that could kill SW480carcinoma cells (HLA-A2 positive/Brachyury high), with minimal lysis ofMCF7 carcinoma (HLA-A2 positive/Brachyury low); (FIG. 3A, donor 07706;FIG. 3B, donor 17663; FIG. 3C, donor 26532).

FIG. 4A is a graph showing that Brachyury-specific T cells from healthydonor PBMCs stimulated with a yeast-Brachyury immunotherapeuticcomposition specifically lyse tumor cells that have the appropriate MHC(SW480, HLA-A2 positive/Brachyury high) versus H226 carcinoma cells(HLA-A2 negative/Brachyury high).

FIG. 4B is a graph showing the expression of Brachyury mRNA relative tothat of a control gene (GAPDH) in the SW480 and H226 tumor cells used inthe experiment shown in FIG. 4A.

FIG. 5 is a graph showing proliferation of CD4⁺ T cells isolated fromthe spleen of mice that were vaccinated with yeast-Brachyury (GI-6301,circles) or control yeast (Yeast control, triangles), in response toindicated doses of purified Brachyury protein or control β-gal protein.

FIG. 6 graph showing that administration of a yeast-Brachyuryimmunotherapeutic composition (GI-6301, circles) of the invention showsa trend towards reducing Brachyury-expressing tumors in mice compared tomice receiving yeast alone (no Brachyury antigen).

FIGS. 7A and 7B are flow cytometry analyses showing that theBrachyury-specific T cell line, T-2-BR-A, binds to a Brachyury-specificHLA-A2 tetramer (FIG. 7B) and not to a control tetramer (FIG. 7A).

FIG. 8 is a flow cytometry analysis showing the expression of perforinin the Brachyury-specific T cell line, T-2-BR-A, after stimulation withBrachyury agonist peptide-pulsed autologous B cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to yeast-based immunotherapeuticcompositions and methods for the prevention and/or treatment of cancersthat express or overexpress Brachyury. The invention includes the use ofa yeast-based immunotherapeutic composition (also referred to asyeast-based immunotherapy) comprising a yeast vehicle and Brachyuryantigens or immunogenic domains thereof (also referred to herein as“yeast-Brachyury immunotherapy” or “yeast-Brachyury immunotherapeuticcompositions”). The inventors describe herein the construction andproduction of novel yeast-Brachyury immunotherapy products, anddemonstrate that yeast-Brachyury immunotherapy expandsBrachyury-specific T cells, including CD8⁺ CTLs, from normal individualsand from cancer patients. In addition, mice immunized withyeast-Brachyury immunotherapeutic compositions generatedBrachyury-specific T cell responses in vivo, and Brachyury-expressingtumor growth was inhibited in these mice. Taken together, the datapresented herein show that yeast-Brachyury immunotherapy is useful forthe elicitation of Brachyury-specific cellular immune responses (CD4⁺and CD8⁺) and for the prevention and treatment of Brachyury-expressingtumors, offering novel therapy for the prevention and/or treatment ofmetastatic cancers and associated conditions.

Yeast-Brachyury immunotherapeutic compositions useful in the presentinvention are uniquely adapted to effectively targetBrachyury-expressing cancers for several reasons. First, Brachyury isinvolved in EMT processes, and therefore, without being bound by theory,the inventors believe that it plays a role in late-stage tumors andmetastatic processes. Accordingly, in one aspect of the invention,yeast-Brachyury immunotherapy is effective at targeting tumor cellsbefore or at the time during which they begin to acquire motility andinvade other tissues, thereby preventing, inhibiting, arresting,reversing or delaying the onset of metastatic cancer and/or theprogression of cancer, and especially metastatic cancer. There is agreat need for effective therapies for late stage cancers, especiallymetastatic cancers, which may have few options for treatment onceconventional cancer therapy has failed. Yeast-Brachyury presents a novelapproach to treat such cancers, or to delay, inhibit, reverse, orprevent them altogether. In addition, yeast-Brachyury immunotherapy canbe used to prevent or delay metastatic cancer or progression of cancerin individuals who have early stage cancer. The therapy is useful, inone embodiment, in cancers that have a high rate of metastaticprogression, and may be useful to arrest progression of the cancer.Furthermore, yeast-Brachyury immunotherapy is useful in individuals whohave a precancerous (pre-malignant) lesion or tumor, in individuals whoare at a high risk for developing a cancer, particularly one that has ahigh rate of metastases, and even in normal individuals as aprophylactic agent for the prevention of cancer, which may be used inconjunction with other prophylactic immunotherapy for cancer, such asdescribed herein.

Yeast-Brachyury immunotherapy also provides a benefit to individuals whoare undergoing other therapy for cancer, including chemotherapy andradiation therapy. More particularly, metastatic cancers are known insome cases to be more resistant to chemotherapy and/or radiation therapythan the primary cancers. Therefore, the yeast-Brachyury immunotherapycompositions of the invention can be used to inhibit or reduce oreliminate chemotherapy resistance or radiation resistance that may occurin metastatic cancer by inhibiting Brachyury expression in the cancer(and thereby inhibiting anti-proliferative influences), and compositionsof the invention may enhance the performance of chemotherapy orradiation therapy in an individual.

Yeast-Brachyury immunotherapy can also be used to treat conditions ordiseases associated with Brachyury expression that may benon-oncological in nature, or that may precede malignant transformation.For example, Brachyury may be upregulated in cells that are infectedwith an infectious agent, e.g., a virus such as Epstein Barr Virus(EBV). Accordingly, yeast-Brachyury immunotherapy can be used to treator prevent any disease or condition associated with Brachyuryexpression, including, but not limited to, infectious diseases, such asviral infection, including, but not limited to, EBV-associatedconditions (e.g., mononucleosis).

Yeast-Brachyury immunotherapy is also readily adaptable to the use ofadditional tumor antigens within the same yeast composition, or to usein combination with other yeast-based immunotherapeutics that targetother tumor antigens (sequentially or concurrently) or otherimmunotherapeutics and treatments/therapies for cancer. Accordingly, theyeast-Brachyury immunotherapy can be adapted to the cancer type, thecancer stage, the cancer grade, the antigens expressed by the tumor, andthe overall medical status of the individual (i.e., the therapy iseasily personalized), and for the individual who already has cancer, itsuse can be modified as cancer progresses in an individual, in order toprovide maximum efficacy at a variety of tumor stages. Yeast-Brachyuryimmunotherapy offers the opportunity to design sophisticated andeffective, individualized approaches for the broad-based prophylacticand/or therapeutic treatment of a wide range of cancers.

Yeast-Brachyury compositions described herein induce innate immuneresponses, as well as adaptive immune responses against the targetantigen (Brachyury), including CD4-dependent TH17 and TH1 T cellresponses and antigen-specific CD8⁺ T cell responses, which includecytotoxic T lymphocyte (CTL) responses, all without the use of exogenousadjuvants, cytokines, or other immunostimulatory molecules, many ofwhich have toxicity issues. In addition, yeast-Brachyuryimmunotherapeutic compositions inhibit regulatory T cell (Treg) numbersand/or functionality, thereby enhancing effector T cell responses thatmight normally be suppressed by the presence of the tumor, for example.Moreover, as compared to immunotherapeutic compositions that immunize bygenerating antibody responses, the antigen-specific, broad-based, andpotent cellular immune responses elicited by yeast-Brachyuryimmunotherapy are believed to be particularly effective in targetingtumor cells. Indeed, numerous studies have shown that immunotherapeuticapproaches are enhanced when tumor cells are targeted via CD8⁺ CTLswhich recognize tumor peptides in the context of MHC Class I molecules.

Yeast-Brachyury immunotherapy is highly adept at activating antigenpresenting cells, and has a unique ability to cross-prime the immuneresponse, generating CD8⁺ CTL responses that are typically effectiveagainst tumors, even in the face of what may otherwise be a suppressiveenvironment. Since this type of immunotherapy utilizes the naturalability of the antigen presenting cell to present relevant immunogens,it is not necessary to know the precise identity of CTL epitopes or MHCClass II epitopes of Brachyury to produce an effective immunotherapeuticaccording to the present invention. In fact, multiple CD4⁺ and CD8⁺ Tcell epitopes can be targeted in a single yeast-Brachyuryimmunotherapeutic composition, and so the yeast-Brachyuryimmunotherapeutics of the invention are not limited to the use of shortpeptides and in fact, the use of longer polypeptides and fusion proteinsin these compositions is efficacious. Accordingly, by usingyeast-Brachyury immunotherapy, the use of algorithms and complexformulas to identify putative T cell epitopes is eliminated.

Furthermore, since Brachyury is not expressed by most normal (non-tumor)tissues, and is typically over-expressed in tumor cells, any “offtarget” effects related to normal tissues are not of concern. Asmentioned above, yeast-Brachyury can be effectively utilized in animmunization protocol (prophylactic or therapeutic) without the use ofexogenous adjuvants, immunostimulatory agents or molecules,costimulatory molecules, or cytokines, although such agents may beincluded, if desired. Moreover, yeast-Brachyury immunotherapy can beadministered repeatedly without losing efficacy, as may be problematicwith other types of immunotherapy.

Compositions of the Invention

One embodiment of the present invention relates to a yeast-basedimmunotherapy composition which can be used to prevent and/or treatcancers characterized by Brachyury expression or overexpression(including cancers that may not contain cells expressing detectableBrachyury initially, but which may or will contain cells expressingBrachyury at later stages of the development of the cancer). Thecomposition is a yeast-Brachyury immunotherapeutic compositioncomprising: (a) a yeast vehicle; and (b) a cancer antigen comprising oneor more Brachyury antigen(s) and/or immunogenic domain(s) thereof. TheBrachyury antigen or immunogenic domain thereof is most typicallyexpressed as a recombinant protein by the yeast vehicle (e.g., by anintact yeast or yeast spheroplast, which can optionally be furtherprocessed to a yeast cytoplast, yeast ghost, or yeast membrane extractor fraction thereof), although it is an embodiment of the invention thatone or more Brachyury antigens are loaded into a yeast vehicle orotherwise complexed with, attached to, mixed with or administered with ayeast vehicle as described herein to form a composition of the presentinvention.

A “yeast-Brachyury immunotherapeutic composition” is a specific type of“yeast-based immunotherapeutic composition” that contains at least oneBrachyury antigen or immunogenic domain thereof. The phrase,“yeast-based immunotherapeutic composition” may be used interchangeablywith “yeast-based immunotherapy product”, “yeast-based immunotherapycomposition”, “yeast-based composition”, “yeast-basedimmunotherapeutic”, “yeast-based vaccine”, or derivatives of thesephrases. An “immunotherapeutic composition” is a composition thatelicits an immune response sufficient to achieve at least onetherapeutic benefit in a subject. As used herein, yeast-basedimmunotherapeutic composition refers to a composition that includes ayeast vehicle component and that elicits an immune response sufficientto achieve at least one therapeutic benefit in a subject. Moreparticularly, a yeast-based immunotherapeutic composition is acomposition that includes a yeast vehicle component and typically, anantigen component, and can elicit or induce an immune response, such asa cellular immune response, including without limitation a Tcell-mediated cellular immune response. In one aspect, a yeast-basedimmunotherapeutic composition useful in the invention is capable ofinducing a CD8⁺ and/or a CD4⁺ T cell-mediated immune response and in oneaspect, a CD8⁺ and a CD4⁺ T cell-mediated immune response, particularlyagainst a target antigen (e.g., a cancer antigen). A CD4⁺ immuneresponse can include TH1 immune responses, TH2 immune responses, TH17immune responses, or any combination of the above. Yeast-basedimmunotherapeutics are particularly capable of generating TH1 and TH17responses. A CD8⁺ immune response can include a cytotoxic T lymphocyte(CTL) response, and yeast-based immunotherapeutics are capable ofgenerating such responses. In one aspect, a yeast-basedimmunotherapeutic composition modulates the number and/or functionalityof regulatory T cells (Tregs) in a subject. Yeast-based immunotherapycan also be modified to promote one type of response over another, e.g.,by the addition of cytokines, antibodies, and/or modulating themanufacturing process for the yeast. Optionally, a yeast-basedimmunotherapeutic composition is capable of eliciting a humoral immuneresponse.

Yeast-Brachyury immunotherapeutic compositions of the invention may beeither “prophylactic” or “therapeutic”. When provided prophylactically,the compositions of the present invention are provided in advance of thedevelopment of, or the detection of the development of, a cancer thatexpresses Brachyury, with the goal of preventing, inhibiting or delayingthe development of Brachyury-expressing tumors; and/or preventing,inhibiting or delaying tumor migration and/or tumor invasion of othertissues (metastases) and/or generally preventing or inhibitingprogression of cancer in an individual. As discussed herein, Brachyuryis expressed in several cancers, including late-stage cancers, and hasbeen shown to be involved in the EMT process, which is a processassociated with invasiveness and migration of tumors, such as inmetastatic cancer. Therefore, prophylactic compositions can beadministered to individuals that appear to be cancer-free (healthy, ornormal, individuals), to individuals with pre-cancerous (pre-malignantlesions), and also to individuals who have cancer, but in whichBrachyury has not yet been detected (i.e. prior to the expression ofBrachyury by tumor cells in the cancer). Individuals who are at highrisk for developing a cancer, particularly a cancer with which Brachyuryexpression and/or metastases are typically associated, may be treatedprophylactically with a composition of the invention. When providedtherapeutically, the immunotherapy compositions are provided to anindividual with a Brachyury-expressing cancer, with the goal ofameliorating the cancer, such as by reducing tumor burden in theindividual; inhibiting tumor growth in the individual; increasingsurvival of the individual; preventing, inhibiting, reversing ordelaying development of tumor migration and/or tumor invasion of othertissues (metastatic cancer) and/or preventing, inhibiting, reversing ordelaying progression of the cancer in the individual. In one aspect,yeast-Brachyury immunotherapy is used therapeutically to inhibit, reduceor eliminate chemotherapy resistance or radiation resistance that mayoccur in metastatic cancer by inhibiting Brachyury expression in thecancer, and compositions of the invention may enhance the performance ofchemotherapy or radiation therapy in an individual.

Typically, a yeast-Brachyury immunotherapy composition includes a yeastvehicle and at least one cancer antigen comprising a Brachyury antigenor immunogenic domain thereof, where the cancer antigen is expressed by,attached to, loaded into, or mixed with the yeast vehicle. In someembodiments, the cancer antigen, Brachyury antigen, or immunogenicdomain thereof is provided as a fusion protein. Several Brachyuryproteins and fusion proteins suitable for use in the compositions andmethods of the invention are described below. In some embodiments, thecancer antigen and the Brachyury antigen are the same element. In someembodiments, the cancer antigen includes other antigens, including othercancer antigens, in addition to the Brachyury antigen. In one aspect ofthe invention, a fusion protein useful as a cancer antigen can includetwo or more antigens, e.g., a Brachyury antigen and another cancerantigen that is not a Brachyury antigen, or two different Brachyuryantigens. In one aspect, the fusion protein can include two or moreimmunogenic domains of one or more antigens, such as two or moreimmunogenic domains of a Brachyury antigen, or two or more epitopes ofone or more antigens, such as two or more epitopes of a Brachyuryantigen.

According to the present invention, a yeast vehicle used in ayeast-Brachyury immunotherapy composition is any yeast cell (e.g., awhole or intact cell) or a derivative thereof (see below) that can beused in conjunction with one or more antigens, immunogenic domainsthereof or epitopes thereof in a composition of the invention (e.g., atherapeutic or prophylactic composition). The yeast vehicle cantherefore include, but is not limited to, a live intact (whole) yeastmicroorganism (i.e., a yeast cell having all its components including acell wall), a killed (dead) or inactivated intact yeast microorganism,or derivatives of intact yeast including: a yeast spheroplast (i.e., ayeast cell lacking a cell wall), a yeast cytoplast (i.e., a yeast celllacking a cell wall and nucleus), a yeast ghost (i.e., a yeast celllacking a cell wall, nucleus and cytoplasm), a subcellular yeastmembrane extract or fraction thereof (also referred to as a yeastmembrane particle and previously as a subcellular yeast particle), anyother yeast particle, or a yeast cell wall preparation.

Yeast spheroplasts are typically produced by enzymatic digestion of theyeast cell wall. Such a method is described, for example, in Franzusoffet al., 1991, Meth. Enzymol. 194, 662-674, incorporated herein byreference in its entirety.

Yeast cytoplasts are typically produced by enucleation of yeast cells.Such a method is described, for example, in Coon, 1978, Natl. CancerInst. Monogr. 48, 45-55 incorporated herein by reference in itsentirety.

Yeast ghosts are typically produced by resealing a permeabilized orlysed cell and can, but need not, contain at least some of theorganelles of that cell. Such a method is described, for example, inFranzusoff et al., 1983, J Biol. Chem. 258, 3608-3614 and Bussey et al.,1979, Biochim. Biophys. Acta 553, 185-196, each of which is incorporatedherein by reference in its entirety.

A yeast membrane particle (subcellular yeast membrane extract orfraction thereof) refers to a yeast membrane that lacks a naturalnucleus or cytoplasm. The particle can be of any size, including sizesranging from the size of a natural yeast membrane to microparticlesproduced by sonication or other membrane disruption methods known tothose skilled in the art, followed by resealing. A method for producingsubcellular yeast membrane extracts is described, for example, inFranzusoff et al., 1991, Meth. Enzymol. 194, 662-674. One may also usefractions of yeast membrane particles that contain yeast membraneportions and, when the antigen or other protein was expressedrecombinantly by the yeast prior to preparation of the yeast membraneparticles, the antigen or other protein of interest. Antigens or otherproteins of interest can be carried inside the membrane, on eithersurface of the membrane, or combinations thereof (i.e., the protein canbe both inside and outside the membrane and/or spanning the membrane ofthe yeast membrane particle). In one embodiment, a yeast membraneparticle is a recombinant yeast membrane particle that can be an intact,disrupted, or disrupted and resealed yeast membrane that includes atleast one desired antigen or other protein of interest on the surface ofthe membrane or at least partially embedded within the membrane.

An example of a yeast cell wall preparation is a preparation of isolatedyeast cell walls carrying an antigen on its surface or at leastpartially embedded within the cell wall such that the yeast cell wallpreparation, when administered to an animal, stimulates a desired immuneresponse against a disease target.

Any yeast strain can be used to produce a yeast vehicle of the presentinvention. Yeast are unicellular microorganisms that belong to one ofthree classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. Oneconsideration for the selection of a type of yeast for use as an immunemodulator is the pathogenicity of the yeast. In one embodiment, theyeast is a non-pathogenic strain such as Saccharomyces cerevisiae. Theselection of a non-pathogenic yeast strain minimizes any adverse effectsto the individual to whom the yeast vehicle is administered. However,pathogenic yeast may be used if the pathogenicity of the yeast can benegated by any means known to one of skill in the art (e.g., mutantstrains). In accordance with one aspect of the present invention,non-pathogenic yeast strains are used.

Genera of yeast strains that may be used in the invention include butare not limited to Saccharomyces, Candida (which can be pathogenic),Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula,Schizosaccharomyces and Yarrowia. In one aspect, yeast genera areselected from Saccharomyces, Candida, Hansenula, Pichia orSchizosaccharomyces, and in one aspect, Saccharomyces is used. Speciesof yeast strains that may be used in the invention include but are notlimited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis,Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcuslaurentii, Cryptococcus neoformans, Hansenula anomala, Hansenulapolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromycesmarxianus var. lactis, Pichia pastoris, Rhodotorula rubra,Schizosaccharomyces pombe, and Yarrowia lipolytica. It is to beappreciated that a number of these species include a variety ofsubspecies, types, subtypes, etc. that are intended to be includedwithin the aforementioned species. In one aspect, yeast species used inthe invention include S. cerevisiae, C. albicans, H polymorpha, P.pastoris and S. pombe. S. cerevisiae is useful as it is relatively easyto manipulate and being “Generally Recognized As Safe” or “GRAS” for useas food additives (GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997).One embodiment of the present invention is a yeast strain that iscapable of replicating plasmids to a particularly high copy number, suchas a S. cerevisiae cir° strain. The S. cerevisiae strain is one suchstrain that is capable of supporting expression vectors that allow oneor more target antigen(s) and/or antigen fusion protein(s) and/or otherproteins to be expressed at high levels. Another yeast strain is usefulin the invention is Saccharomyces cerevisiae W303α. In addition, anymutant yeast strains can be used in the present invention, includingthose that exhibit reduced post-translational modifications of expressedtarget antigens or other proteins, such as mutations in the enzymes thatextend N-linked glycosylation.

The yeast-Brachyury immunotherapy composition of the invention includesat least one cancer antigen comprising a Brachyury antigen. According tothe present invention, the general use herein of the term “antigen”refers: to any portion of a protein (e.g., peptide, partial protein,full-length protein), wherein the protein is naturally occurring orsynthetically derived or designed, to a cellular composition (wholecell, cell lysate or disrupted cells), to an organism (whole organism,lysate or disrupted cells) or to a carbohydrate, or other molecule, or aportion thereof. An antigen may elicit an antigen-specific immuneresponse (e.g., a humoral and/or a cell-mediated immune response)against the same or similar antigens that are encountered by an elementof the immune system (e.g., T cells, antibodies).

An antigen can be as small as a single epitope, a single immunogenicdomain or larger, and can include multiple epitopes or immunogenicdomains. As such, the size of an antigen can be as small as about 8-11amino acids (i.e., a peptide) and as large as: a full length protein, amultimer, a fusion protein, a chimeric protein, a whole cell, a wholemicroorganism, or any portions thereof (e.g., protein fragments(polypeptides) lysates of whole cells or extracts of microorganisms).Antigens useful in the yeast-Brachyury immunotherapeutic of the presentinvention are peptides, polypeptides, full-length proteins, multimers,fusion proteins and chimeric proteins. In addition, antigens can includecarbohydrates, which can be loaded into a yeast vehicle or into acomposition of the invention. It will be appreciated that in someembodiments (e.g., when the antigen is expressed by the yeast vehiclefrom a recombinant nucleic acid molecule), the antigen is a protein,fusion protein, chimeric protein, or fragment thereof, rather than anentire cell or microorganism. For expression in yeast, an antigen is ofa minimum size capable of being expressed recombinantly in yeast if theantigen is the entire protein to be expressed by the yeast, and istypically at least or greater than 25 amino acids in length, or at leastor greater than 26, at least or greater than 27, at least or greaterthan 28, at least or greater than 29, at least or greater than 30, atleast or greater than 31, at least or greater than 32, at least orgreater than 33, at least or greater than 34, at least or greater than35, at least or greater than 36, at least or greater than 37, at leastor greater than 38, at least or greater than 39, at least or greaterthan 40, at least or greater than 41, at least or greater than 42, atleast or greater than 43, at least or greater than 44, at least orgreater than 45, at least or greater than 46, at least or greater than47, at least or greater than 48, at least or greater than 49, or atleast or greater than 50 amino acids in length, or at least or greaterthan 25-50 amino acids in length, or at least or greater than 30-50amino acids in length, or at least or greater than 35-50 amino acids inlength, or at least or greater than 40-50 amino acids in length, or atleast or greater than 45-50 amino acids in length, although smallerproteins may be expressed, and considerably larger proteins (e.g.,hundreds of amino acids in length or even a few thousand amino acids inlength) may be expressed. In one aspect, a full-length protein or aprotein that is lacking between 1 and 20 amino acids from the N- and/orthe C-terminus may be expressed. Fusion proteins and chimeric proteinsare also antigens that may be expressed in the invention. A “targetantigen” is an antigen that is specifically targeted by animmunotherapeutic composition of the invention (i.e., an antigen againstwhich elicitation of an immune response is desired). A “cancer antigen”is an antigen that comprises at least one antigen that is associatedwith a cancer such as an antigen expressed by a tumor cell, such thattargeting the antigen also targets the cancer. A cancer antigen caninclude one or more antigens from one or more proteins, including one ormore tumor-associated proteins. A “Brachyury antigen” is an antigenderived, designed, or produced from a Brachyury protein.

When referring to stimulation of an immune response, the term“immunogen” is a subset of the term “antigen”, and therefore, in someinstances, can be used interchangeably with the term “antigen”. Animmunogen, as used herein, describes an antigen which elicits a humoraland/or cell-mediated immune response (i.e., is immunogenic), such thatadministration of the immunogen to an individual mounts anantigen-specific immune response against the same or similar antigensthat are encountered by the immune system of the individual. In oneembodiment, the immunogen elicits a cell-mediated immune response,including a CD4⁺ T cell response (e.g., TH1, TH2 and/or TH17) and/or aCD8⁺ T cell response (e.g., a CTL response).

An “immunogenic domain” of a given antigen can be any portion, fragmentor epitope of an antigen (e.g., a peptide fragment or subunit or anantibody epitope or other conformational epitope) that contains at leastone epitope that can act as an immunogen when administered to an animal.Therefore, an immunogenic domain is larger than a single amino acid andis at least of a size sufficient to contain at least one epitope thatcan act as an immunogen. For example, a single protein can containmultiple different immunogenic domains. Immunogenic domains need not belinear sequences within a protein, such as in the case of a humoralimmune response, where conformational domains are contemplated.

An epitope is defined herein as a single immunogenic site within a givenantigen that is sufficient to elicit an immune response when provided tothe immune system in the context of appropriate costimulatory signalsand/or activated cells of the immune system. In other words, an epitopeis the part of an antigen that is recognized by components of the immunesystem, and may also be referred to as an antigenic determinant. Thoseof skill in the art will recognize that T cell epitopes are different insize and composition from B cell or antibody epitopes, and that epitopespresented through the Class I MHC pathway differ in size and structuralattributes from epitopes presented through the Class II MHC pathway. Forexample, T cell epitopes presented by Class I MHC molecules aretypically between 8 and 11 amino acids in length, whereas epitopespresented by Class II MHC molecules are less restricted in length andmay be up to 25 amino acids or longer. In addition, T cell epitopes havepredicted structural characteristics depending on the specific MHCmolecules bound by the epitope. Epitopes can be linear sequence epitopesor conformational epitopes (conserved binding regions). Most antibodiesrecognize conformational epitopes.

Brachyury (which may also be referred to as “T”) is a highly conservedprotein among multiple different animal species and is a transcriptionfactor that contains a “T-box” domain or “T-domain”, a DNA-bindingdomain motif shared among several different proteins, collectivelycalled the T-box family of proteins. Human Brachyury was first cloned in1996 (Edwards et al., supra). One nucleotide sequence encoding humanBrachyury is represented herein by SEQ ID NO:1, which is an mRNAsequence that was obtained from GENBANK® Accession No. NM_003181(GI:19743811). SEQ ID NO:1 encodes a 435 amino acid human Brachyuryprotein, the amino acid sequence of which is represented here as SEQ IDNO:2 (also found in GENBANK® Accession No. NP_003172; GI:4507339).

Another human Brachyury protein disclosed herein is a variant of thehuman Brachyury protein represented by SEQ ID NO:2, and has the aminoacid sequence of SEQ ID NO:6. SEQ ID NO:6, also a 435 amino acidprotein, is encoded by a nucleotide sequence represented herein by SEQID NO:5. SEQ ID NO:6 is approximately 99% identical to SEQ ID NO:2 overthe full-length of the protein. SEQ ID NO:6 differs from SEQ ID NO:2 atposition 177 (Asp vs. Gly, respectively), position 368 (Thr vs. Ser,respectively) and position 409 (Asn vs. Asp, respectively).

Another human Brachyury protein disclosed herein is an agonist of thehuman Brachyury protein represented by SEQ ID NO:2 or SEQ ID NO:6. Asgenerally used herein, an “agonist” is any compound or agent, includingwithout limitation small molecules, proteins, peptides, antibodies,nucleic acid binding agents, etc., that binds to a receptor or ligandand produces or triggers a response, which may include agents that mimicor enhance the action of a naturally occurring substance that binds tothe receptor or ligand. When used in the context of a Brachyury antigenof the invention, an “agonist” antigen or protein refers to an antigenor protein that comprises at least one T cell agonist epitope, which mayalso be referred to as a “mimotope”. A mimotope peptide is a peptidethat mimics the structure of a wild-type epitope and as an agonist, themimotope mimics or enhances the action (biological function) of thenatural epitope. For example, the amino acid sequence of SEQ ID NO:12(WLLPGTSTL) is a T cell epitope of a wild-type Brachyury protein. Theamino acid sequence of SEQ ID NO:13 (WLLPGTSTV) is a mimotope or agonistof the T cell epitope of SEQ ID NO:12.

One human Brachyury agonist antigen is represented here by SEQ ID NO:18.SEQ ID NO:18 is a 435 amino acid protein is encoded by a nucleotidesequence represented herein by SEQ ID NO:17. SEQ ID NO:18 is identicalto SEQ ID NO:6, except for a substitution of a leucine at position 254with respect to SEQ ID NO:6 with a valine in SEQ ID NO:18. Thissubstitution creates a T cell agonist epitope in SEQ ID NO:18 atpositions 246 to 254 that, without being bound by theory, is believed toinduce enhanced T cell responses against Brachyury as compared to thewild-type epitope (positions 246 to 254 of SEQ ID NO:6).

Positions 41 to 223 of any of SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:18represent the T-box DNA binding domain of human Brachyury, and the T-boxdomain in other Brachyury sequences, including Brachyury sequences fromother species, can be readily identified by comparison to thesesequences. As used herein, reference to a T-box domain of any Brachyuryprotein described herein or known in the art and utilized in theinvention may include an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive amino acids ofthe Brachyury sequence on the N-terminal and/or the C-terminal end ofthe defined T-box domain (e.g., on either side of positions 41-223 ofSEQ ID NOs:2, 6 or 18). Human Brachyury, including the two humanBrachyury proteins described herein, also contains various CD4⁺ and CD8⁺T cell epitopes. Such epitopes have been described, for example, in WO2008/106551, and include a CD8⁺ CTL epitope, WLLPGTSTL (also referred toherein as Tp2, SEQ ID NO:12), at positions 246 to 254 of SEQ ID NO:2 orSEQ ID NO:6. As discussed above, SEQ ID NO:18 comprises an agonistepitope of SEQ ID NO:12, represented herein by SEQ ID NO:13.

Human Brachyury has very high homology with Brachyury from other animalspecies and therefore, one is able to utilize the sequences of Brachyuryfrom other organisms in the preparation of a yeast-Brachyuryimmunotherapeutic composition of the invention, particularly where thesesequences are identical, substantially homologous, and elicit aneffective immune response against the target antigen (e.g., nativeBrachyury expressed by a tumor cell). For example, murine Brachyury,which was first cloned by Hermann and colleagues in 1990 (Hermann etal., supra) is approximately 85% identical to human Brachyury at thenucleotide level, and approximately 91% identical at the amino acidlevel. With respect to Brachyury from other animals, at the amino acidlevel, human Brachyury is 99.5% identical to Brachyury from Pantroglodytes, 90.1% identical to Brachyury from Canis lupus familiaris,88.5% identical to Brachyury from Bos Taurus, 92.2% identical toBrachyury from Rattus norvegicus, and 80.9% identical to Brachyury fromGallus gallus. Within amino acids 1-223 of Brachyury, which contains theT-box domain, mouse and human Brachyury differ by only two amino acids(at positions 26 and 96). A nucleotide sequence encoding murineBrachyury is represented herein by SEQ ID NO:3, which is an mRNAsequence that was obtained from GENBANK® Accession No. NM_009309(GI:118130357). SEQ ID NO:3 encodes a 436 amino acid murine Brachyuryprotein, the amino acid sequence of which is represented here as SEQ IDNO:4. Positions 41 to 223 of SEQ ID NO:4 represent the T-box DNA bindingdomain of murine Brachyury.

In one embodiment of the invention, a Brachyury antigen comprises orconsists of the amino acid sequence represented by SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:18, or at least one immunogenic domainthereof. In one embodiment, a Brachyury antigen comprises or consists oftwo, three, four, five, or more immunogenic domains of Brachyury. In oneembodiment of the invention, a Brachyury antigen comprises or consistsof the amino acid sequence represented by amino acid positions 1 or 2through one of the last 25 amino acids at the C-terminus of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:18 (i.e., through any one ofpositions 441 to 435 of SEQ ID NO:2 or SEQ ID NO:6 or SEQ ID NO:18, orthrough any one of positions 442 to 436 of SEQ ID NO:4). AnotherBrachyury antigen useful in the invention also includes at least aminoacid positions 1-223 of Brachyury (e.g., positions 1-223 of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:18) or positions 41-223 ofBrachyury (e.g., positions 41-223 of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6 or SEQ ID NO:18). Another Brachyury antigen useful in the inventionincludes from at least amino acid positions 1 to 85 to between position255 and the C-terminus of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQID NO:18. Another Brachyury antigen useful in the invention includesfrom at least amino acid positions 1 to 85 to between position 430 andthe C-terminus of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:18.Another Brachyury antigen useful in the invention includes from at leastamino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to between position255 and the C-terminus of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQID NO:18.

According to any embodiment of the present invention, reference to a“full-length” protein (or a full-length functional domain or full-lengthimmunological domain) includes the full-length amino acid sequence ofthe protein or functional domain or immunological domain, as describedherein or as otherwise known or described in a publicly availablesequence. A protein or domain that is “near full-length”, which is alsoa type of homologue of a protein, differs from a full-length protein ordomain, by the addition or deletion or omission of 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids from the N- and/or C-terminus of such afull-length protein or full-length domain. By way of example, several ofthe fusion proteins described herein comprise a “near full-length”Brachyury antigen since the antigen omits the methionine at position 1and substitutes an N-terminal peptide. General reference to a protein ordomain or antigen can include both full-length and near full-lengthproteins, as well as other homologues thereof.

In one aspect of any embodiments related to a Brachyury antigen, acancer antigen or a Brachyury antigen is of a minimum size sufficient toallow the antigen to be expressed by yeast. For expression in yeast, aprotein is typically at least about 25 amino acids in length, althoughsmaller proteins may be expressed, and considerably larger proteins maybe expressed by yeast. For example, a Brachyury antigen useful in theinvention is a fragment of a Brachyury protein that can be expressedrecombinantly by yeast and that contains at least one immunogenic domainof Brachyury, which could include at least one immunogenic domain of anyof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:18. In one aspect,such an antigen is at least 25 amino acids in length, and contains atleast one immunogenic domain of Brachyury. In one aspect, such anantigen is greater than 30 amino acids in length, and contains at leastone immunogenic domain of Brachyury. In one aspect, such an antigen isat least 25-50 amino acids in length, and contains at least oneimmunogenic domain of Brachyury. In one aspect, such an antigen is atleast 30-50 amino acids in length, and contains at least one immunogenicdomain of Brachyury. In one aspect, such an antigen is at least 35-50amino acids in length, and contains at least one immunogenic domain ofBrachyury. In one aspect, such an antigen is at least 40-50 amino acidsin length, and contains at least one immunogenic domain of Brachyury. Inone aspect, such an antigen is at least 45-50 amino acids in length, andcontains at least one immunogenic domain of Brachyury. In oneembodiment, the Brachyury antigen useful in the present invention is atleast 25 amino acids in length, or at least: 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420,425, or 430 amino acids in length, which can include any fragment of atleast any of these lengths of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 orSEQ ID NO:18.

In one aspect, a Brachyury antigen comprises one or more CTL epitopes,which may include two or more copies of any one, two, three, or more ofthe CTL epitopes described herein. In one aspect, the Brachyury antigencomprises one or more CD4⁺ T cell epitopes. The T cell In one aspect,the Brachyury antigen comprises one or more CTL epitopes and one or moreCD4⁺ T cell epitopes. In one aspect, the T cell epitope is an agonistepitope.

In one aspect, a Brachyury antigen comprises an amino acid sequence ofWLLPGTSTL (SEQ ID NO:12, also represented by positions 245 to 254 of SEQID NO:2 or SEQ ID NO:6). In one aspect, the Brachyury antigen comprisesan amino acid sequence of WLLPGTSTV (SEQ ID NO:13, also represented bypositions 245 to 254 of SEQ ID NO:18). In one aspect, the amino acid atposition 4 of either SEQ ID NO:12 or SEQ ID NO:13 (a proline or P inthese sequences) is substituted with a serine (S), a threonine (T), anisoleucine (I), or a valine (V).

In one aspect, the Brachyury antigen comprises an amino acid sequence ofSQYPSLWSV (SEQ ID NO:14). In one aspect, the amino acid at position 2 ofSEQ ID NO:14 (a glutamine or Q in this sequence) is substituted with aleucine (L). In one aspect, the amino acid at position 4 of SEQ ID NO:14(a proline or P in this sequence) is substituted with a serine (S),threonine (T), leucine (L), or valine (V). In one aspect, the amino acidat position 7 of SEQ ID NO:14 (a tryptophan or W in this sequence) issubstituted with a valine (V), leucine (L), isoleucine (I), serine (S),or threonine (T). In one aspect, the amino acid at position 9 of SEQ IDNO:14 (a valine or V in this sequence) is substituted with a leucine(L). An antigen comprising a sequence having any combination of one ormore of these substitutions in SEQ ID NO:14 is contemplated by theinvention.

In one aspect, the Brachyury antigen comprises an amino acid sequence ofRLIASWTPV (SEQ ID NO:15). In one aspect, the amino acid at position 1 ofSEQ ID NO:15 (an arginine or R in this sequence) is substituted with atyrosine (Y) or a tryptophan (W). In one aspect, the amino acid atposition 6 of SEQ ID NO:15 (a tryptophan or W in this sequence) issubstituted with a valine (V), a lysine (L), an isoleucine (I), a serine(S), or a threonine (T). An antigen comprising a sequence having anycombination of one or both of these substitutions in SEQ ID NO:15 iscontemplated by the invention.

In one aspect, the Brachyury antigen comprises an amino acid sequence ofAMYSFLLDFV (SEQ ID NO:16). In one aspect, the amino acid at position 2of SEQ ID NO:16 (a methionine or M in this sequence) is substituted witha leucine (L).

In one embodiment of the invention, a Brachyury antigen comprises,consists essentially of, or consists of a fusion protein having theamino acid sequence of SEQ ID NO:8. The fusion protein of SEQ ID NO:8 isa single polypeptide with the following sequence elements fused in framefrom N- to C-terminus: (1) an N-terminal peptide to impart resistance toproteasomal degradation and stabilize expression in yeast (positions 1-6of SEQ ID NO:8); (2) a human Brachyury antigen consisting of positions2-435 of SEQ ID NO:6 (positions 7-440 of SEQ ID NO:8); and (3) ahexahistidine tag (positions 441-446 of SEQ ID NO:8). The amino acidsequence of SEQ ID NO:8 is encoded by the polynucleotide sequence of SEQID NO:7.

In another embodiment of the invention, a Brachyury antigen comprises,consists essentially of, or consists of a fusion protein having theamino acid sequence of SEQ ID NO:10. The fusion protein of SEQ ID NO:10is a single polypeptide with the following sequence elements fused inframe from N- to C-terminus: (1) an N-terminal peptide to impartresistance to proteasomal degradation and stabilize expression in yeast(positions 1-6 of SEQ ID NO:10); (2) a murine Brachyury antigenconsisting of positions 2-436 of SEQ ID NO:4 (positions 7-441 of SEQ IDNO:10); and (3) a hexahistidine tag (positions 442-447 of SEQ ID NO:10).The amino acid sequence of SEQ ID NO:10 is encoded by the polynucleotidesequence of SEQ ID NO:9.

In another embodiment of the invention, a Brachyury antigen comprises,consists essentially of, or consists of a fusion protein having theamino acid sequence of SEQ ID NO:20. The fusion protein of SEQ ID NO:20is a single polypeptide with the following sequence elements fused inframe from N- to C-terminus: (1) an N-terminal peptide to impartresistance to proteasomal degradation and stabilize expression(positions 1 to 6 of SEQ ID NO:20, the peptide sequence also representedherein by SEQ ID NO:11); 2) amino acids 2-435 of SEQ ID NO:18 (positions7-440 of SEQ ID NO:20), SEQ ID NO:18 representing a full-length humanBrachyury agonist protein; and (3) a hexahistidine tag (positions441-446 of SEQ ID NO:20). The agonist epitope (SEQ ID NO:13) is locatedat positions 251 to 259 of SEQ ID NO:20 (positions 246 to 254 of SEQ IDNO:18). The amino acid sequence of SEQ ID NO:20 is encoded by thepolynucleotide sequence of SEQ ID NO:19.

A Brachyury antigen useful in the present invention also includesproteins having an amino acid sequence that is at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of any of the Brachyury proteins or antigensdescribed herein over the full length of the protein, or with respect toa defined fragment or domain thereof (e.g., an immunological domain orfunctional domain (domain with at least one biological activity)) thatforms part of the protein. For example, a domain of the Brachyuryprotein described herein includes the T-box domain. An immunologicaldomain has been described in detail above.

In some aspects of the invention, amino acid insertions, deletions,and/or substitutions can be made for one, two, three, four, five, six,seven, eight, nine, ten, or more amino acids of a wild-type or referenceBrachyury protein, provided that the resulting Brachyury protein, whenused as an antigen in a yeast-Brachyury immunotherapeutic composition ofthe invention, elicits an immune response against a native Brachyuryprotein as the wild-type or reference Brachyury protein, which mayinclude an enhanced immune response, a diminished immune response, or asubstantially similar immune response. For example, the inventionincludes the use of Brachyury agonist antigens, which may include one ormore T cell epitopes that have been mutated to enhance the T cellresponse against the Brachyury agonist, such as by improving the avidityor affinity of the epitope for an MHC molecule or for the T cellreceptor that recognizes the epitope in the context of MHC presentation.Brachyury agonists may therefore improve the potency or efficiency of aT cell response against native Brachyury expressed by a tumor cell. TheBrachyury antigen having the amino acid sequence of SEQ ID NO:18 is anon-limiting example of a Brachyury agonist (or a Brachyury antigencomprising an agonist epitope).

In addition, N-terminal expression sequences and the C-terminal tags,such as those described above with respect to the fusion proteins of SEQID NO:8, SEQ ID NO:10, or SEQ ID NO:20 are optional, but may be selectedfrom several different sequences described elsewhere herein to improveor assist with expression, stability, and/or allow for identificationand/or purification of the protein. Also, many different promoterssuitable for use in yeast are known in the art. Furthermore, shortintervening linker sequences (e.g., 1, 2, 3, 4, or 5 amino acidpeptides) may be introduced between portions of a fusion proteincomprising a Brachyury antigen for a variety of reasons, including theintroduction of restriction enzyme sites to facilitate cloning, ascleavage sites for host phagosomal proteases, to accelerate protein orantigen processing, and for future manipulation of the constructs.

Optionally, proteins, including fusion proteins, which are used as acomponent of the yeast-Brachyury immunotherapeutic composition of theinvention are produced using antigen constructs that are particularlyuseful for improving or stabilizing the expression of heterologousantigens in yeast. In one embodiment, the desired antigenic protein(s)or peptide(s) are fused at their amino-terminal end to: (a) a specificsynthetic peptide that stabilizes the expression of the fusion proteinin the yeast vehicle or prevents posttranslational modification of theexpressed fusion protein (such peptides are described in detail, forexample, in U.S. Patent Publication No. 2004-0156858 A1, published Aug.12, 2004, incorporated herein by reference in its entirety); (b) atleast a portion of an endogenous yeast protein, including but notlimited to yeast alpha factor leader sequence, wherein either fusionpartner provides improved stability of expression of the protein in theyeast and/or a prevents post-translational modification of the proteinsby the yeast cells (such proteins are also described in detail, forexample, in U.S. Patent Publication No. 2004-0156858 A1, supra); and/or(c) at least a portion of a yeast protein that causes the fusion proteinto be expressed on the surface of the yeast (e.g., an Aga protein,described in more detail herein). In addition, the present inventionoptionally includes the use of peptides that are fused to the C-terminusof the antigen-encoding construct, particularly for use in the selectionand identification of the protein. Such peptides include, but are notlimited to, any synthetic or natural peptide, such as a peptide tag(e.g., 6× His or hexapeptide) or any other short epitope tag. Peptidesattached to the C-terminus of an antigen according to the invention canbe used with or without the addition of the N-terminal peptidesdiscussed above, and vice versa.

In one embodiment, a synthetic peptide useful in a fusion protein to beexpressed in a yeast is linked to the N-terminus of the antigen, thepeptide consisting of at least two amino acid positions that areheterologous to the antigen, wherein the peptide stabilizes theexpression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein. Thesynthetic peptide and N-terminal portion of the antigen together form afusion protein that has the following requirements: (1) the amino acidresidue at position one of the fusion protein is a methionine (i.e., thefirst amino acid in the synthetic peptide is a methionine); (2) theamino acid residue at position two of the fusion protein is not aglycine or a proline (i.e., the second amino acid in the syntheticpeptide is not a glycine or a proline); (3) none of the amino acidpositions at positions 2-6 of the fusion protein is a methionine (i.e.,the amino acids at positions 2-6, whether part of the synthetic peptideor the protein, if the synthetic peptide is shorter than 6 amino acids,do not include a methionine); and (4) none of the amino acids atpositions 2-6 of the fusion protein is a lysine or an arginine (i.e.,the amino acids at positions 2-6, whether part of the synthetic peptideor the protein, if the synthetic peptide is shorter than 5 amino acids,do not include a lysine or an arginine). The synthetic peptide can be asshort as two amino acids, but in one aspect, is 2-6 amino acids(including 3, 4, 5 amino acids), and can be longer than 6 amino acids,in whole integers, up to about 200 amino acids, 300 amino acids, 400amino acids, 500 amino acids, or more.

In one embodiment, a fusion protein comprises an amino acid sequence ofM-X2-X3-X4-X5-X6, wherein M is methionine; wherein X2 is any amino acidexcept glycine, proline, lysine or arginine; wherein X3 is any aminoacid except methionine, lysine or arginine; wherein X4 is any amino acidexcept methionine, lysine or arginine; wherein X5 is any amino acidexcept methionine, lysine or arginine; and wherein X6 is any amino acidexcept methionine, lysine or arginine. In one embodiment, the X6 residueis a proline. An exemplary synthetic sequence that enhances thestability of expression of an antigen in a yeast cell and/or preventspost-translational modification of the protein in the yeast includes thesequence M-A-D-E-A-P (represented herein by SEQ ID NO:11). In additionto the enhanced stability of the expression product, this fusion partnerdoes not appear to negatively impact the immune response against theimmunizing antigen in the construct. In addition, the synthetic fusionpeptides can be designed to provide an epitope that can be recognized bya selection agent, such as an antibody.

In one aspect of the invention, the yeast vehicle is manipulated suchthat the antigen is expressed or provided by delivery or translocationof an expressed protein product, partially or wholly, on the surface ofthe yeast vehicle (extracellular expression). One method foraccomplishing this aspect of the invention is to use a spacer arm forpositioning one or more protein(s) on the surface of the yeast vehicle.For example, one can use a spacer arm to create a fusion protein of theantigen(s) or other protein of interest with a protein that targets theantigen(s) or other protein of interest to the yeast cell wall. Forexample, one such protein that can be used to target other proteins is ayeast protein (e.g., cell wall protein 2 (cwp2), Aga2, Pir4 or Flo1protein) that enables the antigen(s) or other protein to be targeted tothe yeast cell wall such that the antigen or other protein is located onthe surface of the yeast. Proteins other than yeast proteins may be usedfor the spacer arm; however, for any spacer arm protein, it is mostdesirable to have the immunogenic response be directed against thetarget antigen rather than the spacer arm protein. As such, if otherproteins are used for the spacer arm, then the spacer arm protein thatis used should not generate such a large immune response to the spacerarm protein itself such that the immune response to the targetantigen(s) is overwhelmed. One of skill in the art should aim for asmall immune response to the spacer arm protein relative to the immuneresponse for the target antigen(s). Spacer arms can be constructed tohave cleavage sites (e.g., protease cleavage sites) that allow theantigen to be readily removed or processed away from the yeast, ifdesired. Any known method of determining the magnitude of immuneresponses can be used (e.g., antibody production, lytic assays, etc.)and are readily known to one of skill in the art.

Another method for positioning the target antigen(s) or other proteinsto be exposed on the yeast surface is to use signal sequences such asglycosylphosphatidyl inositol (GPI) to anchor the target to the yeastcell wall. Alternatively, positioning can be accomplished by appendingsignal sequences that target the antigen(s) or other proteins ofinterest into the secretory pathway via translocation into theendoplasmic reticulum (ER) such that the antigen binds to a proteinwhich is bound to the cell wall (e.g., cwp).

In one aspect, the spacer arm protein is a yeast protein. The yeastprotein can consist of between about two and about 800 amino acids of ayeast protein. In one embodiment, the yeast protein is about 10 to 700amino acids. In another embodiment, the yeast protein is about 40 to 600amino acids. Other embodiments of the invention include the yeastprotein being at least 250 amino acids, at least 300 amino acids, atleast 350 amino acids, at least 400 amino acids, at least 450 aminoacids, at least 500 amino acids, at least 550 amino acids, at least 600amino acids, or at least 650 amino acids. In one embodiment, the yeastprotein is at least 450 amino acids in length. Another consideration foroptimizing antigen surface expression, if that is desired, is whetherthe antigen and spacer arm combination should be expressed as a monomeror as dimer or as a trimer, or even more units connected together. Thisuse of monomers, dimers, trimers, etc. allows for appropriate spacing orfolding of the antigen such that some part, if not all, of the antigenis displayed on the surface of the yeast vehicle in a manner that makesit more immunogenic.

Use of yeast proteins can stabilize the expression of fusion proteins inthe yeast vehicle, prevents posttranslational modification of theexpressed fusion protein, and/or targets the fusion protein to aparticular compartment in the yeast (e.g., to be expressed on the yeastcell surface). For delivery into the yeast secretory pathway, exemplaryyeast proteins to use include, but are not limited to: Aga (including,but not limited to, Aga1 and/or Aga2); SUC2 (yeast invertase); alphafactor signal leader sequence; CPY; Cwp2p for its localization andretention in the cell wall; BUD genes for localization at the yeast cellbud during the initial phase of daughter cell formation; Flo1p; Pir2p;and Pir4p.

Other sequences can be used to target, retain and/or stabilize theprotein to other parts of the yeast vehicle, for example, in the cytosolor the mitochondria or the endoplasmic reticulum or the nucleus.Examples of suitable yeast protein that can be used for any of theembodiments above include, but are not limited to, TK, AF, SECT;phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinase PGK andtriose phosphate isomerase TPI gene products for their repressibleexpression in glucose and cytosolic localization; the heat shockproteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whoseproteins are more thermostable upon exposure of cells to heat treatment;the mitochondrial protein CYC1 for import into mitochondria; ACT1.

Methods of producing yeast vehicles and expressing, combining and/orassociating yeast vehicles with antigens and/or other proteins and/oragents of interest to produce yeast-based immunotherapy compositions arecontemplated by the invention.

According to the present invention, the term “yeast vehicle-antigencomplex” or “yeast-antigen complex” is used generically to describe anyassociation of a yeast vehicle with an antigen, and can be usedinterchangeably with “yeast-based immunotherapy composition” when suchcomposition is used to elicit an immune response as described above.Such association includes expression of the antigen by the yeast (arecombinant yeast), introduction of an antigen into a yeast, physicalattachment of the antigen to the yeast, and mixing of the yeast andantigen together, such as in a buffer or other solution or formulation.These types of complexes are described in detail below.

In one embodiment, a yeast cell used to prepare the yeast vehicle istransfected with a heterologous nucleic acid molecule encoding a protein(e.g., the antigen) such that the protein is expressed by the yeastcell. Such a yeast is also referred to herein as a recombinant yeast ora recombinant yeast vehicle. The yeast cell can then be formulated witha pharmaceutically acceptable excipient and administered directly to apatient, stored for later administration, or loaded into a dendriticcell as an intact cell. The yeast cell can also be killed, or it can bederivatized such as by formation of yeast spheroplasts, cytoplasts,ghosts, or subcellular particles, any of which may be followed bystoring, administering, or loading of the derivative into the dendriticcell. Yeast spheroplasts can also be directly transfected with arecombinant nucleic acid molecule (e.g., the spheroplast is producedfrom a whole yeast, and then transfected) in order to produce arecombinant spheroplast that expresses the antigen. Yeast cells or yeastspheroplasts that recombinantly express the antigen(s) may be used toproduce a yeast vehicle comprising a yeast cytoplast, a yeast ghost, ora yeast membrane particle or yeast cell wall particle, or fractionthereof.

In general, the yeast vehicle and antigen(s) and/or other agents can beassociated by any technique described herein. In one aspect, the yeastvehicle was loaded intracellularly with the antigen(s) and/or agent(s).In another aspect, the antigen(s) and/or agent(s) was covalently ornon-covalently attached to the yeast vehicle. In yet another aspect, theyeast vehicle and the antigen(s) and/or agent(s) were associated bymixing. In another aspect, and in one embodiment, the antigen(s) and/oragent(s) are expressed recombinantly by the yeast vehicle or by theyeast cell or yeast spheroplast from which the yeast vehicle wasderived.

A number of antigens and/or other proteins to be produced by a yeastvehicle of the present invention is any number of antigens and/or otherproteins that can be reasonably produced by a yeast vehicle, andtypically ranges from at least one to at least about 6 or more,including from about 2 to about 6 antigens and or other proteins.

Expression of an antigen or other protein in a yeast vehicle of thepresent invention is accomplished using techniques known to thoseskilled in the art. Briefly, a nucleic acid molecule encoding at leastone desired antigen or other protein is inserted into an expressionvector in such a manner that the nucleic acid molecule is operativelylinked to a transcription control sequence in order to be capable ofeffecting either constitutive or regulated expression of the nucleicacid molecule when transformed into a host yeast cell. Nucleic acidmolecules encoding one or more antigens and/or other proteins can be onone or more expression vectors operatively linked to one or moreexpression control sequences. Particularly important expression controlsequences are those which control transcription initiation, such aspromoter and upstream activation sequences. Any suitable yeast promotercan be used in the present invention and a variety of such promoters areknown to those skilled in the art. Promoters for expression inSaccharomyces cerevisiae include, but are not limited to, promoters ofgenes encoding the following yeast proteins: alcohol dehydrogenase I(ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), translational elongation factor EF-1 alpha(TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referredto as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1),galactose-1-phosphate uridyl-transferase (GAL7), UDP-galactose epimerase(GAL10), cytochrome cl (CYC1), Sec7 protein (SECT) and acid phosphatase(PHOS), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10promoters, and including the ADH2/GAPDH promoter, which is induced whenglucose concentrations in the cell are low (e.g., about 0.1 to about 0.2percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise,a number of upstream activation sequences (UASs), also referred to asenhancers, are known. Upstream activation sequences for expression inSaccharomyces cerevisiae include, but are not limited to, the UASs ofgenes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1,ADH2, SUC2, GAL1, GAL7 and GAL10, as well as other UASs activated by theGAL4 gene product, with the ADH2 UAS being used in one aspect. Since theADH2 UAS is activated by the ADR1 gene product, it may be preferable tooverexpress the ADR1 gene when a heterologous gene is operatively linkedto the ADH2 UAS. Transcription termination sequences for expression inSaccharomyces cerevisiae include the termination sequences of theα-factor, GAPDH, and CYC1 genes.

Transcription control sequences to express genes in methyltrophic yeastinclude the transcription control regions of the genes encoding alcoholoxidase and formate dehydrogenase.

Transfection of a nucleic acid molecule into a yeast cell according tothe present invention can be accomplished by any method by which anucleic acid molecule can be introduced into the cell and includes, butis not limited to, diffusion, active transport, bath sonication,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. Transfected nucleic acid molecules can be integrated into ayeast chromosome or maintained on extrachromosomal vectors usingtechniques known to those skilled in the art. Examples of yeast vehiclescarrying such nucleic acid molecules are disclosed in detail herein. Asdiscussed above, yeast cytoplast, yeast ghost, and yeast membraneparticles or cell wall preparations can also be produced recombinantlyby transfecting intact yeast microorganisms or yeast spheroplasts withdesired nucleic acid molecules, producing the antigen therein, and thenfurther manipulating the microorganisms or spheroplasts using techniquesknown to those skilled in the art to produce cytoplast, ghost orsubcellular yeast membrane extract or fractions thereof containingdesired antigens or other proteins.

Effective conditions for the production of recombinant yeast vehiclesand expression of the antigen and/or other protein by the yeast vehicleinclude an effective medium in which a yeast strain can be cultured. Aneffective medium is typically an aqueous medium comprising assimilablecarbohydrate, nitrogen and phosphate sources, as well as appropriatesalts, minerals, metals and other nutrients, such as vitamins and growthfactors. The medium may comprise complex nutrients or may be a definedminimal medium. Yeast strains of the present invention can be culturedin a variety of containers, including, but not limited to, bioreactors,Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates.Culturing is carried out at a temperature, pH and oxygen contentappropriate for the yeast strain. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art (see, forexample, Guthrie et al. (eds.), 1991, Methods in Enzymology, vol. 194,Academic Press, San Diego). For example, under one protocol, liquidcultures containing a suitable medium can be inoculated using culturesobtained from starter plates and/or starter cultures of yeast-Brachyuryimmunotherapy compositions, and are grown for approximately 20 h at 30°C., with agitation at 250 rpm. Primary cultures can then be expandedinto larger cultures as desired. Protein expression from vectors withwhich the yeast were transformed (e.g., Brachyury expression) may beconstitutive if the promoter utilized is a constitutive promoter, or maybe induced by addition of the appropriate induction conditions for thepromoter if the promoter utilized is an inducible promoter (e.g., coppersulfate in the case of the CUP1 promoter). In the case of an induciblepromoter, induction of protein expression may be initiated after theculture has grown to a suitable cell density, which may be at about 0.2Y.U./ml or higher densities.

One non-limiting example of a medium suitable for the culture of ayeast-Brachyury immunotherapy composition of the invention is U2 medium.U2 medium comprises the following components: 20 g/L of glucose, 6.7 g/Lof Yeast nitrogen base containing ammonium sulfate, and 0.04 mg/mL eachof histidine, leucine, tryptophan, and adenine. Another non-limitingexample of a medium suitable for the culture of yeast-Brachyuryimmunotherapy composition of the invention is UL2 medium. UL2 mediumcomprises the following components: 20 g/L of glucose, 6.7 g/L of Yeastnitrogen base containing ammonium sulfate, and 0.04 mg/mL each ofhistidine, tryptophan, and adenine.

In one embodiment of the invention, when an inducible promoter is used(e.g. the CUP1 promoter) to express a Brachyury protein in a yeastvehicle according to the invention, induction of protein expression isinitiated at a higher cell density as compared to the cell density thatwould be suitable for most proteins expressed by yeast using such apromoter. More specifically, the present inventors have discovered thatoptimal Brachyury antigen expression driven by the CUP1 promoter occurswhen the yeast expressing the Brachyury antigen are allowed to grow to acell density of between at least 0.5 Y.U/ml and approximately 2.0Y.U./ml, and in one aspect, to between 0.5 Y.U./ml and approximately 1.5Y.U./ml, and in one aspect, to between at least 1.0 Y.U./ml and about2.0 Y.U./ml, and in another aspect, to at least about 1.0 Y.U./ml, priorto inducing expression of the Brachyury antigen in the yeast. Thepresent inventors have discovered that subsequent to induction ofBrachyury expression, the yeast will double only about 1× to 1.5×.Moreover, after induction of Brachyury expression, the inventors havediscovered that growth of the yeast to cell densities higher than about2.0 Y.U./ml, or for longer than about 6-8 hours, results in decreasedviability of the cultures, while not substantially improving antigenaccumulation in the yeast. Therefore, in one embodiment of theinvention, a yeast-Brachyury immunotherapy composition having antigenexpression under the control of an inducible promoter, such as the CUP1promoter, is grown to mid-log phase prior to inducing antigenexpression. In one aspect, the cells are grown to between about 1 and 2Y.U./ml prior to induction of antigen expression. In one aspect, antigenexpression is induced (e.g., by the addition of copper sulfate) andcontinues for up to 6, 6.5, 7, 7.5, or 8 hours. In one aspect, theinduction occurs at a temperature of about 30° C. and agitation rate of250 rpm.

In some embodiments of the invention, the yeast are grown under neutralpH conditions. As used herein, the general use of the term “neutral pH”refers to a pH range between about pH 5.5 and about pH 8, and in oneaspect, between about pH 6 and about 8. One of skill the art willappreciate that minor fluctuations (e.g., tenths or hundredths) canoccur when measuring with a pH meter. As such, the use of neutral pH togrow yeast cells means that the yeast cells are grown in neutral pH forthe majority of the time that they are in culture. In one embodiment,yeast are grown in a medium maintained at a pH level of at least 5.5(i.e., the pH of the culture medium is not allowed to drop below pH5.5). In another aspect, yeast are grown at a pH level maintained atabout 6, 6.5, 7, 7.5 or 8. The use of a neutral pH in culturing yeastpromotes several biological effects that are desirable characteristicsfor using the yeast as vehicles for immunomodulation. For example,culturing the yeast in neutral pH allows for good growth of the yeastwithout negative effect on the cell generation time (e.g., slowing ofdoubling time). The yeast can continue to grow to high densities withoutlosing their cell wall pliability. The use of a neutral pH allows forthe production of yeast with pliable cell walls and/or yeast that aremore sensitive to cell wall digesting enzymes (e.g., glucanase) at allharvest densities. This trait is desirable because yeast with flexiblecell walls can induce different or improved immune responses as comparedto yeast grown under more acidic conditions, e.g., by promoting thesecretion of cytokines by antigen presenting cells that havephagocytosed the yeast (e.g., TH1-type cytokines including, but notlimited to, IFN-γ, interleukin-12 (IL-12), and IL-2, as well asproinflammatory cytokines such as IL-6). In addition, greateraccessibility to the antigens located in the cell wall is afforded bysuch culture methods. In another aspect, the use of neutral pH for someantigens allows for release of the di-sulfide bonded antigen bytreatment with dithiothreitol (DTT) that is not possible when such anantigen-expressing yeast is cultured in media at lower pH (e.g., pH 5).

In one embodiment, control of the amount of yeast glycosylation is usedto control the expression of antigens by the yeast, particularly on thesurface. The amount of yeast glycosylation can affect the immunogenicityand antigenicity of the antigen, particularly one expressed on thesurface, since sugar moieties tend to be bulky. As such, the existenceof sugar moieties on the surface of yeast and its impact on thethree-dimensional space around the target antigen(s) should beconsidered in the modulation of yeast according to the invention. Anymethod can be used to reduce the amount of glycosylation of the yeast(or increase it, if desired). For example, one could use a yeast mutantstrain that has been selected to have low glycosylation (e.g. mnn1, och1and mnn9 mutants), or one could eliminate by mutation the glycosylationacceptor sequences on the target antigen. Alternatively, one could useyeast with abbreviated glycosylation patterns, e.g., Pichia. One canalso treat the yeast using methods that reduce or alter theglycosylation.

In one embodiment of the present invention, as an alternative toexpression of an antigen or other protein recombinantly in the yeastvehicle, a yeast vehicle is loaded intracellularly with the protein orpeptide, or with carbohydrates or other molecules that serve as anantigen and/or are useful as immunomodulatory agents or biologicalresponse modifiers according to the invention. Subsequently, the yeastvehicle, which now contains the antigen and/or other proteinsintracellularly, can be administered to an individual or loaded into acarrier such as a dendritic cell. Peptides and proteins can be inserteddirectly into yeast vehicles of the present invention by techniquesknown to those skilled in the art, such as by diffusion, activetransport, liposome fusion, electroporation, phagocytosis, freeze-thawcycles and bath sonication. Yeast vehicles that can be directly loadedwith peptides, proteins, carbohydrates, or other molecules includeintact yeast, as well as spheroplasts, ghosts or cytoplasts, which canbe loaded with antigens and other agents after production.Alternatively, intact yeast can be loaded with the antigen and/or agent,and then spheroplasts, ghosts, cytoplasts, or subcellular particles canbe prepared therefrom. Any number of antigens and/or other agents can beloaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4or any whole integer up to hundreds or thousands of antigens and/orother agents, such as would be provided by the loading of amicroorganism or portions thereof, for example.

In another embodiment of the present invention, an antigen and/or otheragent is physically attached to the yeast vehicle. Physical attachmentof the antigen and/or other agent to the yeast vehicle can beaccomplished by any method suitable in the art, including covalent andnon-covalent association methods which include, but are not limited to,chemically crosslinking the antigen and/or other agent to the outersurface of the yeast vehicle or biologically linking the antigen and/orother agent to the outer surface of the yeast vehicle, such as by usingan antibody or other binding partner. Chemical cross-linking can beachieved, for example, by methods including glutaraldehyde linkage,photoaffinity labeling, treatment with carbodiimides, treatment withchemicals capable of linking di-sulfide bonds, and treatment with othercross-linking chemicals standard in the art. Alternatively, a chemicalcan be contacted with the yeast vehicle that alters the charge of thelipid bilayer of yeast membrane or the composition of the cell wall sothat the outer surface of the yeast is more likely to fuse or bind toantigens and/or other agent having particular charge characteristics.Targeting agents such as antibodies, binding peptides, solublereceptors, and other ligands may also be incorporated into an antigen asa fusion protein or otherwise associated with an antigen for binding ofthe antigen to the yeast vehicle.

When the antigen or other protein is expressed on or physically attachedto the surface of the yeast, spacer arms may, in one aspect, becarefully selected to optimize antigen or other protein expression orcontent on the surface. The size of the spacer arm(s) can affect howmuch of the antigen or other protein is exposed for binding on thesurface of the yeast. Thus, depending on which antigen(s) or otherprotein(s) are being used, one of skill in the art will select a spacerarm that effectuates appropriate spacing for the antigen or otherprotein on the yeast surface. In one embodiment, the spacer arm is ayeast protein of at least 450 amino acids. Spacer arms have beendiscussed in detail above.

In yet another embodiment, the yeast vehicle and the antigen or otherprotein are associated with each other by a more passive, non-specificor non-covalent binding mechanism, such as by gently mixing the yeastvehicle and the antigen or other protein together in a buffer or othersuitable formulation (e.g., admixture).

In one embodiment, intact yeast (with or without expression ofheterologous antigens or other proteins) can be ground up or processedin a manner to produce yeast cell wall preparations, yeast membraneparticles or yeast fragments (i.e., not intact) and the yeast fragmentscan, in some embodiments, be provided with or administered with othercompositions that include antigens (e.g., DNA vaccines, protein subunitvaccines, killed or inactivated pathogens, viral vector vaccines) toenhance immune responses. For example, enzymatic treatment, chemicaltreatment or physical force (e.g., mechanical shearing or sonication)can be used to break up the yeast into parts that are used as anadjuvant.

In one embodiment of the invention, yeast vehicles useful in theinvention include yeast vehicles that have been killed or inactivated.Killing or inactivating of yeast can be accomplished by any of a varietyof suitable methods known in the art. For example, heat inactivation ofyeast is a standard way of inactivating yeast, and one of skill in theart can monitor the structural changes of the target antigen, ifdesired, by standard methods known in the art. Alternatively, othermethods of inactivating the yeast can be used, such as chemical,electrical, radioactive or UV methods. See, for example, the methodologydisclosed in standard yeast culturing textbooks such as Methods ofEnzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of theinactivation strategies used should take the secondary, tertiary orquaternary structure of the target antigen into consideration andpreserve such structure as to optimize its immunogenicity.

Yeast vehicles can be formulated into yeast-based immunotherapycompositions or products of the present invention using a number oftechniques known to those skilled in the art. For example, yeastvehicles can be dried by lyophilization. Formulations comprising yeastvehicles can also be prepared by packing yeast in a cake or a tablet,such as is done for yeast used in baking or brewing operations. Inaddition, yeast vehicles can be mixed with a pharmaceutically acceptableexcipient, such as an isotonic buffer that is tolerated by a host orhost cell. Examples of such excipients include water, saline, Ringer'ssolution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.Other useful formulations include suspensions containingviscosity-enhancing agents, such as sodium carboxymethylcellulose,sorbitol, glycerol or dextran. Excipients can also contain minor amountsof additives, such as substances that enhance isotonicity and chemicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer and Tris buffer, while examples of preservatives includethimerosal, m- or o-cresol, formalin and benzyl alcohol. Standardformulations can either be liquid injectables or solids which can betaken up in a suitable liquid as a suspension or solution for injection.Thus, in a non-liquid formulation, the excipient can comprise, forexample, dextrose, human serum albumin, and/or preservatives to whichsterile water or saline can be added prior to administration.

In one embodiment of the present invention, a composition can includeadditional agents, which may also be referred to as biological responsemodifier compounds, or the ability to produce such agents/modifiers. Forexample, a yeast vehicle can be transfected with or loaded with at leastone antigen and at least one agent/biological response modifiercompound, or a composition of the invention can be administered inconjunction with at least one agent/biological response modifier.Biological response modifiers include adjuvants and other compounds thatcan modulate immune responses, which may be referred to asimmunomodulatory compounds, as well as compounds that modify thebiological activity of another compound or agent, such as a yeast-basedimmunotherapeutic, such biological activity not being limited to immunesystem effects. Certain immunomodulatory compounds can stimulate aprotective immune response whereas others can suppress a harmful immuneresponse, and whether an immunomodulatory is useful in combination witha given yeast-based immunotherapeutic may depend, at least in part, onthe disease state or condition to be treated or prevented, and/or on theindividual who is to be treated. Certain biological response modifierspreferentially enhance a cell-mediated immune response whereas otherspreferentially enhance a humoral immune response (i.e., can stimulate animmune response in which there is an increased level of cell-mediatedcompared to humoral immunity, or vice versa.). Certain biologicalresponse modifiers have one or more properties in common with thebiological properties of yeast-based immunotherapeutics or enhance orcomplement the biological properties of yeast-based immunotherapeutics.There are a number of techniques known to those skilled in the art tomeasure stimulation or suppression of immune responses, as well as todifferentiate cell-mediated immune responses from humoral immuneresponses, and to differentiate one type of cell-mediated response fromanother (e.g., a TH17 response versus a TH1 response).

Agents/biological response modifiers useful in the invention mayinclude, but are not limited to, cytokines, chemokines, hormones,lipidic derivatives, peptides, proteins, polysaccharides, small moleculedrugs, antibodies and antigen binding fragments thereof (including, butnot limited to, anti-cytokine antibodies, anti-cytokine receptorantibodies, anti-chemokine antibodies), vitamins, polynucleotides,nucleic acid binding moieties, aptamers, and growth modulators. Somesuitable agents include, but are not limited to, IL-1 or agonists ofIL-1 or of IL-1R, anti-IL-1 or other IL-1 antagonists; IL-6 or agonistsof IL-6 or of IL-6R, anti-IL-6 or other IL-6 antagonists; IL-12 oragonists of IL-12 or of IL-12R, anti-IL-12 or other IL-12 antagonists;IL-17 or agonists of IL-17 or of IL-17R, anti-IL-17 or other IL-17antagonists; IL-21 or agonists of IL-21 or of IL-21R, anti-IL-21 orother IL-21 antagonists; IL-22 or agonists of IL-22 or of IL-22R,anti-IL-22 or other IL-22 antagonists; IL-23 or agonists of IL-23 or ofIL-23R, anti-IL-23 or other IL-23 antagonists; IL-25 or agonists ofIL-25 or of IL-25R, anti-IL-25 or other IL-25 antagonists; IL-27 oragonists of IL-27 or of IL-27R, anti-IL-27 or other IL-27 antagonists;type I interferon (including IFN-α) or agonists or antagonists of type Iinterferon or a receptor thereof; type II interferon (including IFN-γ)or agonists or antagonists of type II interferon or a receptor thereof;anti-CD40, CD40L, lymphocyte-activation gene 3 (LAG3) protein and/orIMP321 (T-cell immunostimulatory factor derived from the soluble form ofLAG3), anti-CTLA-4 antibody (e.g., to release anergic T cells); T cellco-stimulators (e.g., anti-CD137, anti-CD28, anti-CD40); alemtuzumab(e.g., CamPath®), denileukin diftitox (e.g., ONTAK®); anti-CD4;anti-CD25; anti-PD-1, anti-PD-L1, anti-PD-L2; agents that block FOXP3(e.g., to abrogate the activity/kill CD4+/CD25+ T regulatory cells);Flt3 ligand, imiquimod (Aldara™), granulocyte-macrophage colonystimulating factor (GM-CSF); granulocyte-colony stimulating factor(G-CSF), sargramostim (Leukine®); hormones including without limitationprolactin and growth hormone; Toll-like receptor (TLR) agonists,including but not limited to TLR-2 agonists, TLR-4 agonists, TLR-7agonists, and TLR-9 agonists; TLR antagonists, including but not limitedto TLR-2 antagonists, TLR-4 antagonists, TLR-7 antagonists, and TLR-9antagonists; anti-inflammatory agents and immunomodulators, includingbut not limited to, COX-2 inhibitors (e.g., Celecoxib, NSAIDS),glucocorticoids, statins, and thalidomide and analogues thereofincluding IMiD™s (which are structural and functional analogues ofthalidomide (e.g., REVLIMID® (lenalidomide), ACTIMID® (pomalidomide));proinflammatory agents, such as fungal or bacterial components or anyproinflammatory cytokine or chemokine; immunotherapeutic vaccinesincluding, but not limited to, virus-based vaccines, bacteria-basedvaccines, or antibody-based vaccines; and any other immunomodulators,immunopotentiators, anti-inflammatory agents, pro-inflammatory agents,and any agents that modulate the number of, modulate the activationstate of, and/or modulate the survival of antigen-presenting cells or ofTH17, TH1, and/or Treg cells. Any combination of such agents iscontemplated by the invention, and any of such agents combined with oradministered in a protocol with (e.g., concurrently, sequentially, or inother formats with) a yeast-based immunotherapeutic is a compositionencompassed by the invention. Such agents are well known in the art.These agents may be used alone or in combination with other agentsdescribed herein.

Agents can include agonists and antagonists of a given protein orpeptide or domain thereof. As used herein, an “agonist” is any compoundor agent, including without limitation small molecules, proteins,peptides, antibodies, nucleic acid binding agents, etc., that binds to areceptor or ligand and produces or triggers a response, which mayinclude agents that mimic or enhance the action of a naturally occurringsubstance that binds to the receptor or ligand. An “antagonist” is anycompound or agent, including without limitation small molecules,proteins, peptides, antibodies, nucleic acid binding agents, etc., thatblocks or inhibits or reduces the action of an agonist.

Compositions of the invention can further include or can be administeredwith (concurrently, sequentially, or intermittently with) any otheragents or compositions or protocols that are useful for preventing ortreating cancer or any compounds that treat or ameliorate any symptom ofcancer, and particularly cancers associated with Brachyury expression oroverexpression. In addition, compositions of the invention can be usedtogether with other immunotherapeutic compositions, includingprophylactic and/or therapeutic immunotherapy. Indeed, the compositionsof the invention can be used to inhibit or reduce chemotherapyresistance or radiation resistance that may occur in metastatic cancerby inhibiting Brachyury expression in the cancer (and thereby inhibitinganti-proliferative influences) or compositions of the invention mayenhance the performance of chemotherapy or radiation therapy in anindividual. Additional agents, compositions or protocols (e.g.,therapeutic protocols) that are useful for the treatment of cancerinclude, but are not limited to, chemotherapy, surgical resection of atumor, radiation therapy, allogeneic or autologous stem celltransplantation, and/or targeted cancer therapies (e.g., small moleculedrugs, biologics, or monoclonal antibody therapies that specificallytarget molecules involved in tumor growth and progression, including,but not limited to, selective estrogen receptor modulators (SERMs),aromatase inhibitors, tyrosine kinase inhibitors, serine/threoninekinase inhibitors, histone deacetylase (HDAC) inhibitors, retinoidreceptor activators, apoptosis stimulators, angiogenesis inhibitors,poly (ADP-ribose_) polymerase (PARP) inhibitors, or immunostimulators).Any of these additional therapeutic agents and/or therapeutic protocolsmay be administered before, concurrently with, alternating with, orafter the immunotherapy compositions of the invention, or at differenttime points. For example, when given to an individual in conjunctionwith chemotherapy or a targeted cancer therapy, it may be desirable toadminister the yeast-Brachyury immunotherapy compositions during the“holiday” between doses of chemotherapy or targeted cancer therapy, inorder to maximize the efficacy of the immunotherapy compositions.Surgical resection of a tumor may frequently precede administration of ayeast-Brachyury immunotherapy composition, but additional or primarysurgery may occur during or after administration of a yeast-Brachyuryimmunotherapy composition.

The invention also includes a kit comprising any of the compositionsdescribed herein, or any of the individual components of thecompositions described herein. Kits may include additional reagents andwritten instructions or directions for using any of the compositions ofthe invention to prevent or treat cancer associated with Brachyuryexpression or overexpression.

Methods for Administration or Use of Compositions of the Invention

Yeast-Brachyury immunotherapeutic compositions of the invention aredesigned for use to prevent or treat cancers that are associated with orcharacterized by Brachyury expression or overexpression, including bypreventing emergence of such cancers, arresting progression of suchcancers or eliminating such cancers. More particularly, yeast-Brachyuryimmunotherapeutic compositions can be used to prevent, inhibit or delaythe development of Brachyury-expressing tumors, and/or to prevent,inhibit or delay tumor migration and/or tumor invasion of other tissues(metastases) and/or to generally prevent or inhibit progression ofcancer in an individual. Yeast-Brachyury immunotherapeutic compositionscan also be used to ameliorate at least one symptom of the cancer, suchas by reducing tumor burden in the individual; inhibiting tumor growthin the individual; increasing survival of the individual; preventing,inhibiting, reversing or delaying development of tumor migration and/ortumor invasion of other tissues (metastatic cancer) and/or preventing,inhibiting, reversing or delaying progression of the cancer in theindividual. Yeast-Brachyury immunotherapy can also be usedtherapeutically to inhibit, reduce or eliminate chemotherapy resistanceor radiation resistance that may occur in metastatic cancer byinhibiting Brachyury expression in the cancer, and compositions of theinvention may enhance the performance of chemotherapy or radiationtherapy in an individual.

Cancers that are relevant to the compositions and methods of theinvention are any cancer that expresses, or may express, Brachyury, orcancers in proximity to cancers that express or may express Brachyury,and include, but are not limited to, cancer of the breast, smallintestine, stomach, kidney, bladder, uterus, ovary, testes, lung, colon,pancreas, or prostate, and include metastatic and late-stage cancers. Inaddition, Brachyury is expressed in tumors of B cell origin, such aschronic lymphocytic leukemia (CLL), Epstein-Barr virus transformed Bcells, Burkitt's and Hodgkin's lymphomas, as well as metastatic cancersthereof.

One embodiment of the invention relates to a method to inhibit tumormigration and/or to reduce, halt (arrest), reverse or prevent themetastatic progression of cancer in an individual who has cancer, or toreverse the development of metastatic events in a cancer. As discussedabove, Brachyury promotes the epithelial-mesenchymal transition (EMT) inhuman tumor cells, conferring on tumor cells a mesenchymal phenotype, aswell as migratory and invasive abilities, while attenuating tumor cellcycle progression. Therefore, the involvement of Brachyury in metastaticprocesses makes it an ideal target for the prevention or inhibition ofmetastatic processes, including arresting cancer at a pre-metastaticstage. Use of a yeast-Brachyury immunotherapeutic composition of theinvention can be effective to prevent or treat metastatic cancer,including arresting progression of the cancer, in the face of escape (orattempted escape) of the cancer from traditional therapy, such aschemotherapy and radiation. The method includes the steps ofadministering to the individual who has cancer an immunotherapeuticcomposition a yeast-Brachyury immunotherapeutic composition of theinvention as described herein, including, but not limited to: (a) ayeast vehicle; and (b) a cancer antigen comprising at least oneBrachyury antigen.

In one aspect, Brachyury is not detected in the individual's cancer atthe time the composition is first administered. In general, whenBrachyury is not detected in the individual's cancer, the individual mayhave an earlier stage cancer in which Brachyury expression has not yetmanifested (e.g., stage I or stage II), or in which Brachyury expressionis not yet detectable in any event (i.e., Brachyury may or may not beexpressed at a low level or in a small number of tumor cells, but is notyet readily detectable using standard detection methods). In this aspectof the invention, the development of Brachyury-expressing tumor cells isprevented, delayed or inhibited by use of the yeast-Brachyuryimmunotherapeutic composition. As a result, tumor migration and/or othermetastatic processes leading to metastatic progression of the tumor areprevented, delayed or inhibited and/or general arrest of tumorprogression occurs in the individual.

In another aspect, Brachyury expression is or can be detected in theindividual's cancer at the time the composition is first administered.The individual may have stage I, stage II, stage III, or stage IV cancerin this aspect of the invention. In this aspect, use of theyeast-Brachyury immunotherapeutic composition reduces, eliminates orslows or arrests the growth of tumors expressing Brachyury, which canresult in reduction in tumor burden in the individual, inhibition ofBrachyury-expressing tumor growth, and/or increased survival of theindividual. The individual may experience an arrest, slowing or reversalin metastatic processes, improving survival and health of the patient,and furthermore, allowing other therapeutic protocols to treat thecancer.

Indeed, metastatic cancer can be associated with resistance, orincreased resistance, to cancer therapies such as chemotherapy,radiation, or targeted cancer therapy, whereby the cancer “escapes” fromthe therapy or is simply less impacted by the therapy and progresses.Accordingly, there is a need to reduce or eliminate resistance to suchtherapies to improve or enhance the efficacy of the therapy and improvepatient health and survival. Accordingly, one embodiment of theinvention relates to a method to reduce or preventchemotherapy-resistance, targeted cancer therapy-resistance, orradiation-resistance in a patient with cancer. The method comprisesadministering to an individual who has cancer and is receivingchemotherapy and/or radiation therapy for the cancer, a yeast-Brachyuryimmunotherapeutic composition as described herein, which may include acomposition comprising: (a) a yeast vehicle; and (b) a cancer antigencomprising at least one Brachyury antigen. This method of the inventionmay also be used to treat resistance associated with other therapeutictreatments for cancer, including, but not limited to, targeted cancertherapy.

In one aspect of this embodiment, Brachyury is not detected in theindividual's cancer at the time the composition is first administered.In this aspect, administration of a yeast-Brachyury immunotherapeuticcomposition prevents or inhibits the onset of resistance to chemotherapyor radiation therapy by inhibiting the development ofBrachyury-expressing tumor cells in the cancer. In another aspect,Brachyury expression is detected in the individual's cancer at the timethe composition is first administered. In this aspect, the individualmay or may not already be experiencing resistance to chemotherapy orradiation. In either case, administration of the yeast-Brachyuryimmunotherapeutic composition of the invention prevents or inhibits theresistance to chemotherapy or radiation therapy or enhances the abilityof the chemotherapy or radiation therapy to treat the individual, byreducing or eliminating Brachyury-expressing tumor cells in the patient.

Another embodiment of the invention relates to a method to treat cancer,and particularly, a Brachyury-expressing cancer. The method includesadministering to an individual who has a Brachyury-expressing cancer ayeast-Brachyury immunotherapeutic composition described herein, whichcan include a composition comprising: (a) a yeast vehicle; and (b) acancer antigen comprising at least one Brachyury antigen. In one aspect,the method reduces tumor burden in the patient. In one aspect, themethod increases survival of the patient. In one aspect, the methodinhibits tumor growth in the individual. In one aspect, the methodprevents, arrests or reverses metastatic progression of the tumor.

Since Brachyury expression is believed to be more prevalent as a canceradvances or progresses into higher stages (e.g., from stage I to stageII to stage III to stage IV, depending on the particular cancer) and isassociated with metastatic processes, it is an embodiment of theinvention to provide a method to prevent or delay the onset of aBrachyury-expressing cancer, or to arrest the cancer at a pre-metastaticor pre-malignant stage. Such a method includes administering to anindividual in whom Brachyury-expressing cancer cells are not detected ayeast-Brachyury immunotherapeutic composition described herein, whichcan include a composition comprising: (a) a yeast vehicle; and (b) acancer antigen comprising at least one Brachyury antigen. In one aspectof this embodiment, the cancer is known to express or believed to besusceptible to expressing Brachyury at some stage of the cancer in atleast a subset of individuals with the cancer. In one aspect of thisembodiment, the individual already has a cancer, but Brachyury is notdetected in the cancer at the time the composition is firstadministered, meaning that the individual may have an earlier stagecancer in which Brachyury expression has not yet manifested, or in whichBrachyury expression is not yet detectable in any event (i.e., Brachyurymay or may not be expressed at a low level or in a small number of tumorcells, but is not yet readily detectable using standard detectionmethods). In some cases, the type of cancer may be known to have a highrate of metastatic progression, In this aspect, administration of theyeast-Brachyury immunotherapeutic composition prevents, delays orinhibits the development of Brachyury-expressing tumor cells in thepatient's cancer, and therefore prevents, arrests, delays or inhibitsmetastatic processes that accompany Brachyury expression. In anotheraspect, the individual does not have cancer when the composition isadministered. Such an individual may be “predisposed” or likely todevelop cancer, perhaps because of family history or a genetic marker,or because the individual has shown signs of precancerous cells orlesions or has precancerous (premalignant) cells or lesions.

In one aspect, the individual is additionally treated with at least oneother therapeutic compound or therapeutic protocol useful for thetreatment of cancer. Such therapeutic agents and protocols have beendiscussed in detail elsewhere herein. For example, in any of theembodiments regarding methods of the invention described herein, in oneaspect, when the individual has cancer (regardless of the status ofdetectable Brachyury expression in tumor cells) the individual is beingtreated or has been treated with another therapy for cancer. Suchtherapy can include any of the therapeutic protocols or use of anytherapeutic compound or agent described previously herein, including,but not limited to, chemotherapy, radiation therapy, targeted cancertherapy, surgical resection of a tumor, stem cell transfer, cytokinetherapy, adoptive T cell transfer, and/or administration of a secondimmunotherapeutic composition. In the case of administration of a secondimmunotherapeutic composition, such compositions may include, but arenot limited to, additional yeast-based immunotherapy, recombinantvirus-based immunotherapy (viral vectors), cytokine therapy,immunostimulant therapy (including chemotherapy with immunostimulatingproperties), DNA vaccines, and other immunotherapy compositions.

In one aspect, the second immunotherapeutic composition includes asecond cancer antigen that does not include Brachyury antigen. Forexample, a second immunotherapeutic composition useful in combinationwith a yeast-Brachyury immunotherapeutic composition is ayeast-immunotherapeutic composition comprising another cancer antigen.Such cancer antigens may include, but are not limited to,carcinoembryonic antigen (CEA), point mutated Ras oncoprotein, MUC-1,EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, normal andpoint mutated p53 oncoproteins, PSMA, tyrosinase, TRP-1 (gp75),NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2, hTERT,p73, B-RAF, adenomatous polyposis coli (APC), Myc, von Hippel-Lindauprotein (VHL), Rb-1, Rb-2, androgen receptor (AR), Smad4, MDR1, Flt-3,BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H, HERV-K, TWIST, Mesothelin,NGEP, modifications of such antigens, splice variants of such antigens,and epitope agonists of such antigens, as well as combinations of suchantigens, and/or immunogenic domains thereof, modifications thereof,variants thereof, and/or epitope agonists thereof.

As used herein, to “treat” a cancer, or any permutation thereof (e.g.,“treated for cancer”, etc.) generally refers to administering acomposition of the invention once the cancer has occurred (e.g., oncethe cancer has been diagnosed or detected in an individual), with atleast one therapeutic goal of the treatment (as compared to in theabsence of this treatment) including: reduction in tumor burden,inhibition of tumor growth, increase in survival of the individual,delaying, inhibiting, arresting or preventing the onset or developmentof metastatic cancer (such as by delaying, inhibiting, arresting orpreventing the onset of development of tumor migration and/or tumorinvasion of tissues outside of primary cancer and/or other processesassociated with metastatic progression of cancer), delaying or arrestingcancer progression, improvement of immune responses against the tumor,improvement of long term memory immune responses against the tumorantigens, and/or improved general health of the individual. To “prevent”or “protect” from a cancer, or any permutation thereof (e.g.,“prevention of cancer”, etc.), generally refers to administering acomposition of the invention before a cancer has occurred, or before aspecific stage of cancer or tumor antigen expression in a cancer hasoccurred (e.g., before Brachyury expression is detected in the cancer),with at least one goal of the treatment (as compared to in the absenceof this treatment) including: preventing or delaying the onset ordevelopment of a cancer, or, should the cancer occur after thetreatment, at least reducing the severity of the cancer (e.g., reducingthe level of tumor growth, arresting cancer progression, improving theimmune response against the cancer, inhibiting metastatic processes) orimproving outcomes in the individual (e.g., improving survival).

The present invention includes the delivery (administration,immunization) of a yeast-Brachyury immunotherapeutic composition of theinvention to a subject or individual. The administration process can beperformed ex vivo or in vivo, but is typically performed in vivo. Exvivo administration refers to performing part of the regulatory stepoutside of the patient, such as administering a composition of thepresent invention to a population of cells (dendritic cells) removedfrom a patient under conditions such that a yeast vehicle, antigen(s)and any other agents or compositions are loaded into the cell, andreturning the cells to the patient. The therapeutic composition of thepresent invention can be returned to a patient, or administered to apatient, by any suitable mode of administration.

Administration of a composition can be systemic, mucosal and/or proximalto the location of the target site (e.g., near a site of a tumor).Suitable routes of administration will be apparent to those of skill inthe art, depending on the type of cancer to be prevented or treatedand/or the target cell population or tissue. Various acceptable methodsof administration include, but are not limited to, intravenousadministration, intraperitoneal administration, intramuscularadministration, intranodal administration, intracoronary administration,intraarterial administration (e.g., into a carotid artery), subcutaneousadministration, transdermal delivery, intratracheal administration,intraarticular administration, intraventricular administration,inhalation (e.g., aerosol), intracranial, intraspinal, intraocular,aural, intranasal, oral, pulmonary administration, impregnation of acatheter, and direct injection into a tissue. In one aspect, routes ofadministration include: intravenous, intraperitoneal, subcutaneous,intradermal, intranodal, intramuscular, transdermal, inhaled,intranasal, oral, intraocular, intraarticular, intracranial, andintraspinal. Parenteral delivery can include intradermal, intramuscular,intraperitoneal, intrapleural, intrapulmonary, intravenous,subcutaneous, atrial catheter and venal catheter routes. Aural deliverycan include ear drops, intranasal delivery can include nose drops orintranasal injection, and intraocular delivery can include eye drops.Aerosol (inhalation) delivery can also be performed using methodsstandard in the art (see, for example, Stribling et al., Proc. Natl.Acad. Sci. USA 189:11277-11281, 1992). In one aspect, a yeast-Brachyuryimmunotherapeutic composition of the invention is administeredsubcutaneously. In one aspect, the yeast-Brachyury immunotherapeuticcomposition is administered directly into a tumor milieu.

In general, a suitable single dose of a yeast-Brachyuryimmunotherapeutic composition is a dose that is capable of effectivelyproviding a yeast vehicle and the Brachyury antigen to a given celltype, tissue, or region of the patient body in an amount effective toelicit an antigen-specific immune response against one or more Brachyuryantigens or epitopes, when administered one or more times over asuitable time period. For example, in one embodiment, a single dose of ayeast-Brachyury of the present invention is from about 1×10⁵ to about5×10⁷ yeast cell equivalents per kilogram body weight of the organismbeing administered the composition. In one aspect, a single dose of ayeast vehicle of the present invention is from about 0.1 Yeast Units(Y.U., which is 1×10⁶ yeast cells or yeast cell equivalents) to about100 Y.U. (1×10⁹ cells) per dose (i.e., per organism), including anyinterim dose, in increments of 0.1×10⁶ cells (i.e., 1.1×10⁶, 1.2×10⁶,1.3×10⁶ . . . ). In one embodiment, a suitable dose includes dosesbetween 1 Y.U. and 40 Y.U. and in one aspect, between 10 Y.U. and 40Y.U. In one embodiment, the doses are administered at different sites onthe individual but during the same dosing period. For example, a 40 Y.U.dose may be administered by injecting 10 Y.U. doses to four differentsites on the individual during one dosing period. The invention includesadministration of an amount of the yeast-Brachyury immunotherapycomposition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 Y.U. or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredifferent sites on an individual to form a single dose. One Yeast Unit(Y.U.) is 1×10⁷ yeast cells or yeast cell equivalents.

“Boosters” or “boosts” of a therapeutic composition are administered,for example, when the immune response against the antigen has waned oras needed to provide an immune response or induce a memory responseagainst a particular antigen or antigen(s). Boosters can be administeredabout 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, or monthly, bimonthly,quarterly, annually, and/or in a few or several year increments afterthe original administration, depending on the status of the individualbeing treated and the goal of the therapy at the time of administration(e.g., prophylactic, active treatment, maintenance). In one embodiment,an administration schedule is one in which doses of yeast-Brachyuryimmunotherapeutic composition is administered at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more times over a time period of from weeks, to months,to years. In one embodiment, the doses are administered weekly orbiweekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed bybiweekly or monthly doses as needed to achieve the desired preventativeor therapeutic treatment for cancer. Additional boosters can then begiven at similar or longer intervals (months or years) as a maintenanceor remission therapy, if desired.

In one aspect of the invention, one or more additional therapeuticagents or therapeutic protocols are administered or performedsequentially and/or concurrently with the administration of theyeast-Brachyury immunotherapy composition (e.g., surgical resection ofthe tumor, administration of chemotherapy, administration of radiationtherapy, administration of another immunotherapy composition orprotocol, cytokine therapy, adoptive T cell transfer, or stem celltransplantation). For example, one or more therapies can be administeredor performed prior to the first dose of yeast-Brachyury immunotherapycomposition or after the first dose is administered. In one embodiment,one or more therapies can be administered or performed in an alternatingmanner with the dosing of yeast-Brachyury immunotherapy composition,such as in a protocol in which the yeast-Brachyury composition isadministered at prescribed intervals in between one or more consecutivedoses of chemotherapy or other therapy. In one embodiment, theyeast-Brachyury immunotherapy composition is administered in one or moredoses over a period of time prior to commencing additional therapies. Inother words, the yeast-Brachyury immunotherapeutic composition isadministered as a monotherapy for a period of time, and then anadditional therapy is added (e.g., chemotherapy), either concurrentlywith new doses of yeast-Brachyury immunotherapy, or in an alternatingfashion with yeast-Brachyury immunotherapy. Alternatively or inaddition, another therapy may be administered for a period of time priorto beginning administration of the yeast-Brachyury immunotherapycomposition, and the concepts may be combined (e.g., surgical resectionof a tumor, followed by monotherapy with yeast-Brachyury immunotherapyfor several weeks, followed by alternating doses of chemotherapy andyeast-Brachyury immunotherapy for weeks or months, optionally followedby monotherapy using yeast-Brachyury immunotherapy or another therapy,or by a new protocol of combinations of therapy provided sequentially,concurrently, or in alternating fashion). Various protocols for thetreatment of cancer using yeast-Brachyury immunotherapy are contemplatedby the invention, and these examples should be considered to benon-limiting examples of various possible protocols.

In one aspect of the invention, additional antigens other than Brachyuryare also targeted using yeast-based immunotherapy, in addition totargeting Brachyury. Such additional target antigens can be includedwithin the same yeast-vehicle as the Brachyury antigens, or additionalyeast-based immunotherapy compositions targeting different antigens canbe produced and then combined as desired depending on the individual tobe treated, the antigens expressed by the type of cancer or by theindividual's particular tumor, and/or depending on the stage of cancerin the individual, or the stage of treatment of the individual. Forexamples a combination of antigens may be selected that cover: (1)antigens involved in seminal events in cancer development, such asmutated Ras, antigens involved in or associated with dysregulation ofcellular processes, such as CEA, and (3) Brachyury, which is involved inmetastatic processes. For example, on or more other yeast-basedimmunotherapy compositions may express one or more antigens including,but not limited to, carcinoembryonic antigen (CEA), point mutated Rasoncoprotein, MUC-1, EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100,MUC-2, normal and point mutated p53 oncoproteins, PSMA, tyrosinase,TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA,HER-2/neu/c-erb/B2, hTERT, p73, B-RAF, adenomatous polyposis coli (APC),Myc, von Hippel-Lindau protein (VHL), Rb-1, Rb-2, androgen receptor(AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H,HERV-K, TWIST, Mesothelin, NGEP, modifications of such antigens, splicevariants of such antigens, and epitope agonists of such antigens, aswell as combinations of such antigens, and/or immunogenic domainsthereof, modifications thereof, variants thereof, and/or epitopeagonists thereof. One, two, three, or more of these yeast-basedimmunotherapy compositions may be administered to an individual priorto, concurrently or alternating with, and/or after administration of ayeast-Brachyury immunotherapy composition, in order to optimizetargeting of antigens in the individual's tumor. As above, additionaltherapies can also be used in such protocols (e.g., surgical resectionof tumor, chemotherapy, targeted cancer therapy, radiation therapy,etc.).

In one embodiment of the invention, a method to treat cancer isprovided. The method includes the steps of: (a) administering to anindividual who has cancer in which Brachyury expression has not beendetected, a first immunotherapeutic composition comprising a yeastvehicle and a first cancer antigen that does not comprise a Brachyuryantigen; and (b) administering to the individual, prior to, concurrentlywith, or subsequent to, administration of the first immunotherapeuticcomposition a second immunotherapeutic composition comprising a yeastvehicle and a second cancer antigen comprising a Brachyury antigen. Inadditional embodiments, the method can include administering one or moreadditional immunotherapeutic compositions, wherein the each of the oneor more additional immunotherapeutic compositions comprises anadditional cancer antigen. The additional antigen can be any of thoseknown in the art or described herein, including, but not limited to,mutated Ras, carcinoembryonic antigen (CEA), and MUC-1.

In another embodiment of the invention, a method to treat cancerincludes the following steps: (a) administering to an individual who hascancer a first immunotherapeutic composition comprising a yeast vehicleand a mutated Ras antigen; (b) administering to the individual of (a) asecond immunotherapeutic composition comprising a yeast vehicle and anantigen selected from the group consisting of carcinoembryonic antigen(CEA) and mucin-1 (MUC-1); and (c) administering to the individual of(a) and (b) a third immunotherapeutic composition comprising a yeastvehicle and a Brachyury antigen. One or more of the steps ofadministration in (a), (b) and (c) can be performed concurrently, orsequentially. Steps may be repeated as needed to treat a particularindividual's cancer, and the cancer antigens can be modified before orduring treatment to specifically address the particular individual'scancer.

In the method of the present invention, compositions and therapeuticcompositions can be administered to animal, including any vertebrate,and particularly to any member of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Livestock include mammals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Mammals to treat or protectutilizing the invention include humans, non-human primates, dogs, cats,mice, rats, goats, sheep, cattle, horses and pigs.

An “individual” is a vertebrate, such as a mammal, including withoutlimitation a human. Mammals include, but are not limited to, farmanimals, sport animals, pets, primates, mice and rats. The term“individual” can be used interchangeably with the term “animal”,“subject” or “patient”.

General Techniques Useful in the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,nucleic acid chemistry, and immunology, which are well known to thoseskilled in the art. Such techniques are explained fully in theliterature, such as, Methods of Enzymology, Vol. 194, Guthrie et al.,eds., Cold Spring Harbor Laboratory Press (1990); Biology and activitiesof yeasts, Skinner, et al., eds., Academic Press (1980); Methods inyeast genetics: a laboratory course manual, Rose et al., Cold SpringHarbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle andCell Biology, Pringle et al., eds., Cold Spring Harbor Laboratory Press(1997); The Yeast Saccharomyces: Gene Expression, Jones et al., eds.,Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces:Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds.,Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Harlow and Lane (1988), Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York; Harlow and Lane(1999) Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (jointly referred to hereinas “Harlow and Lane”), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000);Casarett and Doull's Toxicology The Basic Science of Poisons, C.Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin, W.Orenstein, and P. Offit, eds., Fifth Edition (2008).

GENERAL DEFINITIONS

A “TARMOGEN®” (Globelmmune, Inc., Louisville, Colo.) generally refers toa yeast vehicle expressing one or more heterologous antigensextracellularly (on its surface), intracellularly (internally orcytosolically) or both extracellularly and intracellularly. TARMOGEN®shave been generally described (see, e.g., U.S. Pat. No. 5,830,463).Certain yeast-based immunotherapy compositions, and methods of makingand generally using the same, are also described in detail, for example,in U.S. Pat. No. 5,830,463, U.S. Pat. No. 7,083,787, U.S. Pat. No.7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu et al., CancerResearch 64:5084-5088 (2004), and in Bernstein et al., Vaccine 2008 Jan.24; 26(4):509-21, each of which is incorporated herein by reference inits entirety.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another compound but differs slightly incomposition (as in the replacement of one atom by an atom of a differentelement or in the presence of a particular functional group, or thereplacement of one functional group by another functional group). Thus,an analog is a compound that is similar or comparable in function andappearance, but has a different structure or origin with respect to thereference compound.

The terms “substituted”, “substituted derivative” and “derivative”, whenused to describe a compound, means that at least one hydrogen bound tothe unsubstituted compound is replaced with a different atom or achemical moiety.

Although a derivative has a similar physical structure to the parentcompound, the derivative may have different chemical and/or biologicalproperties than the parent compound. Such properties can include, butare not limited to, increased or decreased activity of the parentcompound, new activity as compared to the parent compound, enhanced ordecreased bioavailability, enhanced or decreased efficacy, enhanced ordecreased stability in vitro and/or in vivo, and/or enhanced ordecreased absorption properties.

In general, the term “biologically active” indicates that a compound(including a protein or peptide) has at least one detectable activitythat has an effect on the metabolic, physiological, chemical, or otherprocesses of a cell, a tissue, or an organism, as measured or observedin vivo (i.e., in a natural physiological environment) or in vitro(i.e., under laboratory conditions).

According to the present invention, the term “modulate” can be usedinterchangeably with “regulate” and refers generally to upregulation ordownregulation of a particular activity. As used herein, the term“upregulate” can be used generally to describe any of: elicitation,initiation, increasing, augmenting, boosting, improving, enhancing,amplifying, promoting, or providing, with respect to a particularactivity. Similarly, the term “downregulate” can be used generally todescribe any of: decreasing, reducing, inhibiting, ameliorating,diminishing, lessening, blocking, or preventing, with respect to aparticular activity.

In one embodiment of the present invention, any of the amino acidsequences described herein can be produced with from at least one, andup to about 20, additional heterologous amino acids flanking each of theC- and/or N-terminal ends of the specified amino acid sequence. Theresulting protein or polypeptide can be referred to as “consistingessentially of” the specified amino acid sequence. According to thepresent invention, the heterologous amino acids are a sequence of aminoacids that are not naturally found (i.e., not found in nature, in vivo)flanking the specified amino acid sequence, or that are not related tothe function of the specified amino acid sequence, or that would not beencoded by the nucleotides that flank the naturally occurring nucleicacid sequence encoding the specified amino acid sequence as it occurs inthe gene, if such nucleotides in the naturally occurring sequence weretranslated using standard codon usage for the organism from which thegiven amino acid sequence is derived. Similarly, the phrase “consistingessentially of”, when used with reference to a nucleic acid sequenceherein, refers to a nucleic acid sequence encoding a specified aminoacid sequence that can be flanked by from at least one, and up to asmany as about 60, additional heterologous nucleotides at each of the 5′and/or the 3′ end of the nucleic acid sequence encoding the specifiedamino acid sequence. The heterologous nucleotides are not naturallyfound (i.e., not found in nature, in vivo) flanking the nucleic acidsequence encoding the specified amino acid sequence as it occurs in thenatural gene or do not encode a protein that imparts any additionalfunction to the protein or changes the function of the protein havingthe specified amino acid sequence.

According to the present invention, the phrase “selectively binds to”refers to the ability of an antibody, antigen-binding fragment orbinding partner of the present invention to preferentially bind tospecified proteins. More specifically, the phrase “selectively binds”refers to the specific binding of one protein to another (e.g., anantibody, fragment thereof, or binding partner to an antigen), whereinthe level of binding, as measured by any standard assay (e.g., animmunoassay), is statistically significantly higher than the backgroundcontrol for the assay. For example, when performing an immunoassay,controls typically include a reaction well/tube that contain antibody orantigen binding fragment alone (i.e., in the absence of antigen),wherein an amount of reactivity (e.g., non-specific binding to the well)by the antibody or antigen-binding fragment thereof in the absence ofthe antigen is considered to be background. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA, immunoblot assays, etc.).

General reference to a protein or polypeptide used in the presentinvention includes full-length proteins, near full-length proteins(defined above), or any fragment, domain (structural, functional, orimmunogenic), conformational epitope, or a homologue or variant of agiven protein. A fusion protein may also be generally referred to as aprotein or polypeptide. An isolated protein, according to the presentinvention, is a protein (including a polypeptide or peptide) that hasbeen removed from its natural milieu (i.e., that has been subject tohuman manipulation) and can include purified proteins, partiallypurified proteins, recombinantly produced proteins, and syntheticallyproduced proteins, for example. As such, “isolated” does not reflect theextent to which the protein has been purified. Preferably, an isolatedprotein of the present invention is produced recombinantly. According tothe present invention, the terms “modification” and “mutation” can beused interchangeably, particularly with regard to themodifications/mutations to the amino acid sequence of proteins orportions thereof (or nucleic acid sequences) described herein.

As used herein, the term “homologue” or “variant” is used to refer to aprotein or peptide which differs from a reference protein or peptide(i.e., the “prototype” or “wild-type” protein) by minor modifications tothe reference protein or peptide, but which maintains the basic proteinand side chain structure of the naturally occurring form. Such changesinclude, but are not limited to: changes in one or a few amino acid sidechains; changes one or a few amino acids, including deletions (e.g., atruncated version of the protein or peptide) insertions and/orsubstitutions; changes in stereochemistry of one or a few atoms; and/orminor derivatizations, including but not limited to: methylation,glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue or variant can have enhanced,decreased, or substantially similar properties as compared to thereference protein or peptide. A homologue or variant can include anagonist of a protein or an antagonist of a protein. Homologues orvariants can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated reference protein, direct proteinsynthesis, or modifications to the nucleic acid sequence encoding theprotein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis, resulting in the encoding of aprotein variant. In addition, naturally occurring variants of areference protein may exist (e.g., isoforms, allelic variants, or othernatural variants that may occur from individual to individual) and maybe isolated, produced and/or utilized in the invention.

A homologue or variant of a given protein may comprise, consistessentially of, or consist of, an amino acid sequence that is at leastabout 45%, or at least about 50%, or at least about 55%, or at leastabout 60%, or at least about 65%, or at least about 70%, or at leastabout 75%, or at least about 80%, or at least about 85%, or at leastabout 86% identical, or at least about 87% identical, or at least about88% identical, or at least about 89% identical, or at least about 90%,or at least about 91% identical, or at least about 92% identical, or atleast about 93% identical, or at least about 94% identical, or at leastabout 95% identical, or at least about 96% identical, or at least about97% identical, or at least about 98% identical, or at least about 99%identical (or any percent identity between 45% and 99%, in whole integerincrements), to the amino acid sequence of the reference protein (e.g.,an amino acid sequence specified herein, or the amino acid sequence of aspecified protein). In one embodiment, the homologue or variantcomprises, consists essentially of, or consists of, an amino acidsequence that is less than 100% identical, less than about 99%identical, less than about 98% identical, less than about 97% identical,less than about 96% identical, less than about 95% identical, and so on,in increments of 1%, to less than about 70% identical to the amino acidsequence of the reference protein.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a Basic Local Alignment Search Tool (BLAST) basic homology searchusing blastp for amino acid searches and blastn for nucleic acidsearches with standard default parameters, wherein the query sequence isfiltered for low complexity regions by default (such as described inAltschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.” Nucleic Acids Res.25:3389-3402, incorporated herein by reference in its entirety); (2) aBLAST alignment of two sequences (e.g., using the parameters describedbelow); (3) and/or PSI-BLAST with the standard default parameters(Position-Specific Iterated BLAST. It is noted that due to somedifferences in the standard parameters between Basic BLAST and BLAST fortwo sequences, two specific sequences might be recognized as havingsignificant homology using the BLAST program, whereas a search performedin Basic BLAST using one of the sequences as the query sequence may notidentify the second sequence in the top matches. In addition, PSI-BLASTprovides an automated, easy-to-use version of a “profile” search, whichis a sensitive way to look for sequence homologues. The program firstperforms a gapped BLAST database search. The PSI-BLAST program uses theinformation from any significant alignments returned to construct aposition-specific score matrix, which replaces the query sequence forthe next round of database searching. Therefore, it is to be understoodthat percent identity can be determined by using any one of theseprograms.

Two specific sequences can be aligned to one another using BLAST asdescribed in Tatusova and Madden, (1999), “Blast 2 sequences—a new toolfor comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250, incorporated herein by reference in its entirety. Such asequence alignment is performed in blastp or blastn using the BLAST 2.0algorithm to perform a Gapped BLAST search (BLAST 2.0) between the twosequences allowing for the introduction of gaps (deletions andinsertions) in the resulting alignment. For purposes of clarity herein,a BLAST sequence alignment for two sequences is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

An isolated nucleic acid molecule is a nucleic acid molecule that hasbeen removed from its natural milieu (i.e., that has been subject tohuman manipulation), its natural milieu being the genome or chromosomein which the nucleic acid molecule is found in nature. As such,“isolated” does not necessarily reflect the extent to which the nucleicacid molecule has been purified, but indicates that the molecule doesnot include an entire genome or an entire chromosome or a segment of thegenome containing more than one gene, in which the nucleic acid moleculeis found in nature. An isolated nucleic acid molecule can include acomplete gene. An isolated nucleic acid molecule that includes a gene isnot a fragment of a chromosome that includes such gene, but ratherincludes the coding region and regulatory regions associated with thegene, but no additional genes that are naturally found on the samechromosome. An isolated nucleic acid molecule may also include portionsof a gene. An isolated nucleic acid molecule can also include aspecified nucleic acid sequence flanked by (i.e., at the 5′ and/or the3′ end of the sequence) additional nucleic acids that do not normallyflank the specified nucleic acid sequence in nature (i.e., heterologoussequences). Isolated nucleic acid molecule can include DNA, RNA (e.g.,mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although thephrase “nucleic acid molecule” primarily refers to the physical nucleicacid molecule and the phrase “nucleic acid sequence” primarily refers tothe sequence of nucleotides on the nucleic acid molecule, the twophrases can be used interchangeably, especially with respect to anucleic acid molecule, or a nucleic acid sequence, being capable ofencoding a protein or domain of a protein.

A recombinant nucleic acid molecule is a molecule that can include atleast one of any nucleic acid sequence encoding any one or more proteinsdescribed herein operatively linked to at least one of any transcriptioncontrol sequence capable of effectively regulating expression of thenucleic acid molecule(s) in the cell to be transfected. Although thephrase “nucleic acid molecule” primarily refers to the physical nucleicacid molecule and the phrase “nucleic acid sequence” primarily refers tothe sequence of nucleotides on the nucleic acid molecule, the twophrases can be used interchangeably, especially with respect to anucleic acid molecule, or a nucleic acid sequence, being capable ofencoding a protein. In addition, the phrase “recombinant molecule”primarily refers to a nucleic acid molecule operatively linked to atranscription control sequence, but can be used interchangeably with thephrase “nucleic acid molecule” which is administered to an animal.

A recombinant nucleic acid molecule includes a recombinant vector, whichis any nucleic acid sequence, typically a heterologous sequence, whichis operatively linked to the isolated nucleic acid molecule encoding afusion protein of the present invention, which is capable of enablingrecombinant production of the fusion protein, and which is capable ofdelivering the nucleic acid molecule into a host cell according to thepresent invention. Such a vector can contain nucleic acid sequences thatare not naturally found adjacent to the isolated nucleic acid moleculesto be inserted into the vector. The vector can be either RNA or DNA,either prokaryotic or eukaryotic, and preferably in the presentinvention, is a plasmid useful for transfecting yeast. Recombinantvectors can be used in the cloning, sequencing, and/or otherwisemanipulating of nucleic acid molecules, and can be used in delivery ofsuch molecules (e.g., as in a DNA composition or a viral vector-basedcomposition). Recombinant vectors are preferably used in the expressionof nucleic acid molecules, and can also be referred to as expressionvectors. Preferred recombinant vectors are capable of being expressed ina transfected host cell, such as a yeast.

In a recombinant molecule of the present invention, nucleic acidmolecules are operatively linked to expression vectors containingregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the host cell and thatcontrol the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude nucleic acid molecules that are operatively linked to one ormore expression control sequences. The phrase “operatively linked”refers to linking a nucleic acid molecule to an expression controlsequence in a manner such that the molecule is expressed whentransfected (i.e., transformed, transduced or transfected) into a hostcell.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as algae, bacteria andyeast. In microbial systems, the term “transformation” is used todescribe an inherited change due to the acquisition of exogenous nucleicacids by the microorganism and is essentially synonymous with the term“transfection.” Therefore, transfection techniques include, but are notlimited to, transformation, chemical treatment of cells, particlebombardment, electroporation, microinjection, lipofection, adsorption,infection and protoplast fusion.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following example describes the production of a yeast-Brachyuryimmunotherapeutic composition.

In this experiment, yeast (Saccharomyces cerevisiae) were engineered toexpress human Brachyury under the control of the copper-induciblepromoter, CUP1, or the constitutive promoter, TEF2, producingyeast-Brachyury immunotherapy compositions. In each case, a fusionprotein comprising a Brachyury antigen was produced as a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:8 (1) an N-terminal peptide toimpart resistance to proteasomal degradation and stabilize expression(positions 1 to 6 of SEQ ID NO:8, the peptide sequence also representedherein by SEQ ID NO:11); 2) amino acids 2-435 of SEQ ID NO:6, SEQ IDNO:6 representing a near full-length human Brachyury protein (positions7-440 of SEQ ID NO:8); and (3) a hexahistidine tag (positions 441-446 ofSEQ ID NO:8). The amino acid sequences used in this fusion protein canbe modified by the use of additional or alternate amino acids flankingeither end of the Brachyury antigen, if desired, and shorter portions ofthe Brachyury antigen may also be used. A nucleic acid sequence encodingthe fusion protein of SEQ ID NO:8 (codon optimized for yeast expression)is represented herein by SEQ ID NO:7.

Briefly, DNA encoding a full length human Brachyury protein from aBrachyury-PCRII plasmid provided by the National Cancer Institute (Dr.Jeffrey Schlom) was amplified using PCR, and then inserted at EcoRI andSpeI cloning sites behind the CUP1 promoter (vector pGI-100) or the TEF2promoter (vectors plu011 or pGI-172) in yeast 2 μm expression vectors.Nucleotide sequences encoding the N-terminal stabilization peptide,MADEAP (SEQ ID NO:11) and a C-terminal hexahistidine peptide were alsoadded to the plasmid vector to encode the complete fusion proteinrepresented by SEQ ID NO:8. The resulting plasmids were transformed intoDH5a for plasmid storage, and into Saccharomyces cerevisiae W303α forproduction of the yeast-Brachyury immunotherapeutic compositions.

Transformation into Saccharomyces cerevisiae was performed by lithiumacetate/polyethylene glycol transfection, and primary transfectants wereselected on solid minimal plates lacking Uracil (UDM; uridine dropoutmedium). Colonies were selected by growing in U2 (uridine dropoutmedium) or UL2 (uridine and leucine dropout medium) medium at 30° C.

The yeast-Brachyury immunotherapy composition comprising apolynucleotide encoding the human Brachyury fusion protein representedby SEQ ID NO:8 under the control of the CUP1 promoter is also referredto herein as GI-6301. The yeast-Brachyury immunotherapy compositioncomprising a polynucleotide encoding the human Brachyury fusion proteinrepresented by SEQ ID NO:8 under the control of the TEF2 promoter (invector plu011) is also referred to herein as GI-6302. The yeastBrachyury immunotherapy composition comprising a polynucleotide encodingthe human Brachyury fusion protein represented by SEQ ID NO:8 under thecontrol of the TEF2 promoter (in vector pGI-172) is also referred toherein as GI-6303.

Liquid cultures lacking uridine (U2) or lacking uridine and leucine(UL2) were inoculated using the plates and starter cultures describedabove, and were grown for 20 h at 30° C., 250 rpm. pH buffered mediacontaining 4.2 g/L of Bis-Tris (BT-U2; BT-UL2) were also inoculated toevaluate yeast-Brachyury immunotherapeutics produced under neutral pHmanufacturing conditions (data not shown). Primary cultures were used toinoculate final cultures of the same formulation.

Recipe for U2 Liquid Media:

15 g/L of glucose

6.7 g/L of Yeast nitrogen base containing ammonium sulfate

0.04 g/L each of histidine, tryptophan, adenine and 0.06 g/L of leucine

Recipe for UL2 Liquid Media:

15 g/L of glucose

6.7 g/L of Yeast nitrogen base containing ammonium sulfate

0.04 g/L each of histidine, tryptophan, and adenine

In initial experiments comparing yeast-Brachyury immunotherapeuticcompositions under the control of different promoters, CUP1-driven(inducible expression) yeast-Brachyury expression was initiated by theaddition of 0.5 mM copper sulfate after the yeast-Brachyury culturereached a density of approximately 0.2 Y.U./ml, and was continued untilthe culture reached a density of 0.5-1.5 Y.U. (yeast-Brachyury doubledonly about 1-1.5 after the addition of copper sulfate, but a largeamount of Brachyury protein was produced by the cells). TEF2-drivenyeast-Brachyury expression is constitutive, and growth of these cellswas continued until the cultures reached a density of between 1.1 to 4.0Y.U./ml. The cells from each culture were then harvested, washed andheat-killed at 56° C. for 1 hour in PBS. Live cells from each culturewere also processed for comparison.

After heat-kill of the cultures, the cells were washed three times inPBS. Total protein expression was measured by a TCAprecipitation/nitrocellulose binding assay and by Western blot using ananti-his tag monoclonal antibody and an anti-Brachyury antibody (Abcam,Cambridge, Mass.). Protein content was quantified usingsemi-quantitative digital imaging methods.

The results of the initial expression experiments (data not shown)demonstrated that each of the yeast-Brachyury immunotherapy compositionsof the invention expressed the Brachyury fusion protein, i.e., usingeither the CUP1 promoter or the TEF2 promoter, and expression wasdetected using either media (U2 and UL2). In addition, antigenexpression was detectable in both heat-killed and live yeast cells (datanot shown). Brachyury expression was significantly higher in theyeast-Brachyury immunotherapeutic composition comprising the CUP1promoter (GI-6301) and so this composition was selected for furtherstudies, including expression optimization and for in vitro and in vivoexperiments (see Examples below).

FIG. 1A shows expression of Brachyury in GI-6301 using both U2 and UL2media using the anti-Brachyury antibody for detection. Control yeastexpressing a non-Brachyury antigen did not stain with the antibody. FIG.1B shows expression of Brachyury in the same GI-6301 preparations, usinganti-His to identify the hexahistidine tag on the Brachyury fusionprotein. Control yeast expressing a non-Brachyury antigen but having ahexahistidine tag is also shown. These results showed good Brachyuryexpression using either media, although expression in UL2 media wassignificantly higher.

Example 2

The following example describes the identification of conditions forantigen expression and manufacturing of the yeast-Brachyuryimmunotherapeutic composition, GI-6301.

To determine the optimum density for copper induction of GI-6301 antigenexpression, starter and intermediate cultures of GI-6301 were preparedusing the standard growth conditions in UL2 media described in Example 1above. Aliquots of the culture were then diluted to 0.5 Y.U./ml, 1.0Y.U./ml and 1.5 Y.U./ml and incubated at 30° C. for 1 hour. 0.5 mM CuSO₄was added to the cultures to induce Brachyury expression, and culturingwas continued. Cells were collected and counted at 6 hours and 14 hoursfor measurement of cell density. 20 Y.U. of heat-killed yeast from eachcondition was lysed, total protein was measured, and Western blots weregenerated using anti-His.

TABLE 1 Induction Time 0 hours 6 hours 14 hours Cell Density 0.5 1.030.96 (Y.U./ml) 1.0 1.88 1.74 1.5 3.14 2.7 

As shown in Table 1, yeast only doubled about 1 time after copperinduction (other experiments showed up to 1.5× doubling), and celldensity and viability (not shown) decreased after 6 hours of copperinduction. FIG. 2 shows that all three induction densities resulted insignificant expression of Brachyury, with a trend toward higherBrachyury expression at the higher induction densities. However,additional experiments using induction starting densities of 2.1 Y.U./mland 2.8 Y.U./ml and 375 μM CuSO₄ showed that protein expression began todecrease as the density of the cultures at the start of copper inductionincreased, and did not significantly improve after about 6-8 hours (datanot shown).

Next, the effect of the amount of CuSO₄ on Brachyury expression wasinvestigated. GI-6301 was grown from starter and intermediate culturesin UL2 media as described in Example 1. Aliquots of the culture werethen diluted to 1.0 Y.U./ml and incubated at 30° C. for 1 hour. CuSO₄was added to each culture at a concentration of either 375 μM or 500 μM,and induction of protein expression was allowed to proceed to varioustime points (2 hrs, 4 hrs, 6 hrs, 8 hrs, 24 hrs), at which point thecells were harvested, heat-killed, and processed for evaluation ofprotein expression using anti-His Western blots as described above.While both concentrations of CuSO₄ resulted in good expression ofBrachyury, protein expression using 375 μM appeared to be slightlybetter, particularly at later time points (data not shown).

Accordingly, for CUP1-driven yeast-Brachyury (inducible expression), theinventors discovered that induction of antigen expression at mid-logphase growth of the yeast was optimal for antigen production. Forproduction of the yeast-Brachyury immunotherapeutic composition(GI-6301) used in the following Examples, cells were grown in UL2 mediaas described in Example 1 to between 1 and 2 Y.U./ml, and were theninduced by the addition of 0.375-0.5 mM copper sulfate for up to 6-8hours at 30° C., 250 rpm. Cells were harvested, washed and heat killedat 56° C. for 1 h in PBS.

Example 3

The following example describes the construction and production of anadditional yeast-Brachyury immunotherapeutic composition, where theBrachyury antigen contains a T cell agonist epitope.

In this experiment, yeast (Saccharomyces cerevisiae) were engineered toexpress a human Brachyury antigen that is a near-full-length Brachyuryprotein comprising the T cell epitope WLLPGTSTV (SEQ ID NO:13), which isan agonist epitope. The native Brachyury T cell epitope, present in SEQID NO:6 or 8, for example, is WLLPGTSTL (SEQ ID NO:12). The humanBrachyury agonist antigen was expressed under the control of thecopper-inducible promoter, CUP1, producing a yeast-Brachyuryimmunotherapy composition. More particularly, a fusion proteincomprising a Brachyury agonist antigen (i.e., a Brachyury antigencontaining at least one agonist epitope) was produced as a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:20 (1) an N-terminal peptide toimpart resistance to proteasomal degradation and stabilize expression(positions 1 to 6 of SEQ ID NO:20, the peptide sequence also representedherein by SEQ ID NO:11); 2) amino acids 2-435 of SEQ ID NO:18(represented by positions 7-440 of SEQ ID NO:20; SEQ ID NO:18 representsa full-length human Brachyury agonist protein having a single amino acidsubstitution at position 254 as compared to wild-type Brachyuryprotein); and (3) a hexahistidine tag (positions 441-446 of SEQ IDNO:20). The agonist epitope (SEQ ID NO:13) is located at positions 251to 259 of SEQ ID NO:20 (positions 246 to 254 of SEQ ID NO:18). The aminoacid sequences used in this fusion protein can be modified by the use ofadditional or alternate amino acids flanking either end of the Brachyuryantigen, if desired, and shorter portions of the Brachyury antigen mayalso be used. A nucleic acid sequence encoding the fusion protein of SEQID NO:20 (codon optimized for yeast expression) is represented herein bySEQ ID NO:19.

Briefly, DNA encoding the near full-length human Brachyury protein asdescribed in Example 1 (i.e., full-length Brachyury minus the N-terminalmethionine), modified by site directed mutagenesis to introduce asubstitution of a valine for the leucine at position 254 with respect tothe full-length Brachyury protein, was amplified using PCR, and theninserted at EcoRI and SpeI cloning sites behind the CUP1 promoter(vector pGI-100) in yeast 2 μm expression vectors. Nucleotide sequencesencoding the N-terminal stabilization peptide, MADEAP (SEQ ID NO:11) anda C-terminal hexahistidine peptide were also added to the plasmid vectorto encode the complete fusion protein represented by SEQ ID NO:20. Theresulting plasmids were transformed into DH5α for plasmid storage, andinto Saccharomyces cerevisiae W303α for production of theyeast-Brachyury immunotherapeutic composition.

Transformation into Saccharomyces cerevisiae was performed by lithiumacetate/polyethylene glycol transfection, and primary transfectants wereselected on solid minimal plates lacking Uracil (UDM; uridine dropoutmedium). Colonies were selected by growing in UL2 (uridine and leucinedropout medium) medium at 30° C.

The yeast-Brachyury immunotherapy composition comprising apolynucleotide encoding the human Brachyury agonist fusion proteinrepresented by SEQ ID NO:20 under the control of the CUP1 promoter isalso referred to herein as GI-6305.

GI-6305 cells were grown in UL2 media as described in Example 1 tobetween 1 and 2 Y.U./ml, and were then induced by the addition of0.375-0.5 mM copper sulfate for up to 6-8 hours at 30° C., 250 rpm,using the conditions developed by the inventors for GI-6301 as describedin Example 2. Cells were harvested, washed and heat killed at 56° C. for1 h in PBS.

After heat-kill of the cultures, the cells were washed three times inPBS. Total protein expression was measured by a TCAprecipitation/nitrocellulose binding assay and by Western blot using ananti-his tag monoclonal antibody and an anti-Brachyury antibody (Abcam,Cambridge, Mass.). Protein content was quantified usingsemi-quantitative digital imaging methods.

FIG. 1C shows the robust expression of Brachyury agonist antigen inGI-6305 using anti-His to identify the hexahistidine tag on theBrachyury fusion protein. The approximate antigen content for GI-6305grown in UL2 medium in this experiment was >22615 ng/Y.U.

Example 4

The following example demonstrates the expansion of Brachyury-specific Tcells using a yeast-Brachyury immunotherapeutic composition of theinvention.

To determine whether T cells from normal donors were capable ofgenerating T cells that are specific for Brachyury antigen, dendriticcells (DCs) were prepared from the peripheral blood mononuclear cells(PBMCs) of two normal donors. Briefly, isolated PBMCs were cultured for5-days in the presence of GM-CSF and IL-4, and were subsequentlyincubated with Control Yeast (also denoted “YVEC”, which is aSaccharomyces cerevisiae yeast that is transformed with an empty vector,or vector that does not contain an antigen-encoding insert) or BrachyuryYeast (GI-6301, described in Examples 1 and 2 above), at a ratio ofyeast:DCs=1:1. After 48-hours co-culture, the DCs were used as APCs forstimulation of autologous T cells. Each cycle of stimulation, designatedas IVS (in vitro stimulation), consisted of 3 days culture in absence ofIL-2, following by 4 additional days in the presence of recombinant IL-2(20 U/ml). At the end of IVS 2, T cells were stained with a controltetramer or a tetramer specific for the Brachyury peptide Tp2(WLLPGTSTL, positions 246 to 254 of SEQ ID NO:2 or SEQ ID NO:6). Table 2shows the percentage of CD8+ T cells that stained positive with eachtetramer.

TABLE 2 Control Brachyury Donor Stimulation Tetramer Tetramer 07706Control Yeast 0.21 0.30 Brachyury Yeast 0.28 0.67 17663 Control Yeast0.04 0.29 Brachyury Yeast 0.05 0.54

In a second experiment, dendritic cells (DCs) were prepared from PBMCsof nine normal donors using a 5-day culture in presence of GM-CSF andIL-4, subsequently incubated with Brachyury yeast (GI-6301), at a ratioyeast:DCs=1:1, as described above. After 48-hours in co-culture, the DCswere used as APCs for stimulation of autologous T cells. Each cycle ofIVS was performed as described above. At the end of IVS 2, T cells werestained with a control tetramer or a tetramer specific for the Brachyurypeptide Tp2. Table 3 shows the percentage of CD8+ T cells that stainedpositive with each Tetramer.

TABLE 3 Control Brachyury Donor Stimulation Tetramer Tetramer 07706Brachyury Yeast 0.28 0.67 17663 Brachyury Yeast 0.05 0.54 32249Brachyury Yeast 0.01 1.24 29004 Brachyury Yeast 0.02 0.36 19063Brachyury Yeast 0.10 2.57 06852 Brachyury Yeast 0.05 0.33 26532Brachyury Yeast 0.07 0.11 12172 Brachyury Yeast 0.01 0.11 26725Brachyury Yeast 0.01 0.20

The results in Tables 2 and 3 show that stimulation of normal donor Tcells with a yeast-Brachyury immunotherapeutic of the inventionincreases the percentage of tetramer-positive CD8⁺ T cells in a majorityof the normal donors, as compared to controls, indicating that normalhuman T cells have the capacity to recognize Brachyury as an immunogen.

Example 5

The following example demonstrates the ability of a yeast-Brachyuryimmunotherapeutic composition to generate Brachyury-specific CTLs fromnormal donor PBMCs that lyse Brachyury-expressing targets.

In this experiment, Brachyury-specific T cells from three of the normaldonors from Table 2 above were expanded in vitro using DCs incubatedwith Brachyury yeast (GI-6301) for 2 cycles of IVS (as described inExample 4). A third IVS was carried out with DCs matured in presence ofCD40L and pulsed with the Brachyury-specific Tp2 peptide (WLLPGTSTL,positions 246 to 254 of SEQ ID NO:2 or SEQ ID NO:6). At day 5, CD8⁺ Tcells were isolated and used in an overnight cytotoxic T lymphocyte(CTL) assay against SW480 (HLA-A2⁺/Brachyury high) and MCF7(HLA-A2⁺/Brachyury low) tumor cell targets, at the indicatedeffector:target (ET) ratios (see FIG. 3). Shown in Table 4 is thepercentage of CD8⁺ T cells that stained positive with a control tetramerversus a Brachyury-specific Tp2 tetramer.

TABLE 4 Normal Control Brachyury Donor Stimulation Tetramer Tetramer07706 Brachyury 0.33 1.84 Yeast/Tp2 17663 Brachyury 0.11 0.65 Yeast/Tp226532 Brachyury 0.05 0.11 Yeast/Tp2

FIGS. 3A (donor 07706), 3B (donor 17663) and 3C (donor 26532) show thatPBMCs from two out of three normal donors were capable of generatingCD8⁺ CTLs that could kill targets expressing Brachyury. Taken together,these data demonstrate that yeast-Brachyury immunotherapeuticcompositions can generate Brachyury-specific CTLs that are capable ofkilling a Brachyury-expressing tumor cell.

In order to show that yeast-Brachyury immunotherapy can induceBrachyury-specific CTLs in the absence of pulsing with a specificpeptide (i.e., by generating CTLs against potentially multiple differentCTL epitopes), additional experiments were performed using normal donorT cells expanded in vitro using only the yeast-Brachyuryimmunotherapeutic composition, GI-6301 (i.e., no peptide pulse).Briefly, Brachyury-specific T cells from normal donor PBMCs (donor19063) were expanded in vitro by using DCs incubated with GI-6301 for 2cycles of IVS (as described in Example 4). At day 5, CD8⁺ T cells wereisolated and used in an overnight CTL assay against SW480 (HLA-A2positive/Brachyury high) and H226 (HLA-A2 negative/Brachyury high) tumorcells, at an effector:target (ET) ratio of 15:1. FIG. 4A shows thepercentage of specific lysis of SW480 and H226 tumor cells. FIG. 4Bshows the expression of Brachyury mRNA relative to that of GAPDH inSW480 and H226 tumor cells by real-time RT-PCR. These experimentsfurther demonstrate that yeast-Brachyury immunotherapeutic compositioncan generate Brachyury-specific CTLs that are capable of killing aBrachyury-expressing tumor cell.

Example 6

The following example demonstrates that a yeast-Brachyury composition ofthe invention can expand Brachyury-specific T cells from cancerpatients.

In this experiment, DCs were prepared from the PBMCs of two breastcancer patients, post-vaccination with viral vector vaccines comprisingCEA and MUC-1 antigens. The DCs were prepared in a 5-day culture inpresence of GM-CSF and IL-4 as described in Example 4, followed byincubation in presence of Brachyury yeast (GI-6301) at a ratio ofyeast:DCs=1:1. After 48-hours co-culture, the DCs were used as APCs forstimulation of autologous T cells. Each cycle of IVS consisted of 3 daysin absence of IL-2, following by 4 additional days in presence of 20U/ml of recombinant IL-2. Shown in Table 5 is the percentage of CD8⁺ Tcells (IVS1) that stained positive with a control tetramer or a tetramerspecific for the Brachyury peptide Tp2.

TABLE 5 Control Brachyury Patient Stimulation Tetramer Tetramer BreastPt 01 Brachyury Yeast 0.11 0.42 Breast Pt 10 Brachyury Yeast 0.23 0.91

The results in Table 5 demonstrate that stimulation of T cells frombreast cancer donors with a yeast-Brachyury immunotherapeutic of theinvention increases the percentage of tetramer-positive CD8⁺ T cells ina majority of the donors, as compared to controls, indicating that Tcells from donors with ongoing cancer have the capacity to recognizeBrachyury as an immunogen.

Example 7

The following Example demonstrates the generation of CD4⁺ T cellresponses specific for Brachyury in vivo using yeast-Brachyuryimmunotherapy.

In this experiment, C57BL/6 mice were vaccinated weekly for a total of 4times with 4 YU of yeast-hBrachyury (GI-6301), administered at fourseparate injection sites at 1 YU per site). Two weeks after the finalboost, the mice were sacrificed and CD4⁺ T cells were purified andassayed for proliferation in presence of various concentrations ofBrachyury purified protein (obtained from insect cells). As a control,n-Gal was used at 40 μg/ml.

The results showing the proliferation of CD4⁺ T cells isolated from thespleens of animals vaccinated with yeast-control (YVEC, see Example 4)and yeast-hBrachyury (GI-6301) are shown in FIG. 5. FIG. 5 shows thatimmunization with yeast-Brachyury (GI-6301) generates Brachyury-specificCD4⁺ T cells.

Example 8

The following example demonstrates that immunization withyeast-Brachyury immunotherapeutic composition reducesBrachyury-expressing tumors in vivo.

In this experiment, C57BL/6 mice received 1×10⁶ MC38-phBrachyury cells(MC38 tumor cells expressing a recombinant human Brachyury) via the tailvein (day 0). Four days post-tumor implantation, animals began receivingweekly vaccinations with yeast control (YVEC, see Example 4) versusyeast-hBrachyury (GI-6301), administered at a dose of 1YU per site atfour different sites (4YU total per dose). At day 40 post-tumorimplantation, animals were sacrificed and the number of lung tumornodules was evaluated. Results from two combined experiments are shownin FIG. 6. Table 6 shows the mean lung tumor number (±SEM) and thenumber (and percentage) of animals bearing≧5 lung nodules.

TABLE 6 Lung Tumors Animals Bearing ≧ 5 Lung Vaccine Treatment (mean ±SEM) Nodules (%) Yeast-Control (YVEC) 4.1 ± 1.2 7/15 (46.7%)Yeast-Brachyury (GI-6301) 1.9 ± 0.5 2/15 (13.3%)

The results in FIG. 6 and Table 6 demonstrate that administration of ayeast-Brachyury immunotherapeutic composition of the invention iscapable of reducing Brachyury-expressing tumors in mice, as compared tomice receiving yeast alone (no Brachyury antigen).

Example 9

The following example demonstrates the generation of Brachyury-specificCD4⁺ T cell responses in vitro using yeast-Brachyury immunotherapy inhuman peripheral blood mononuclear cells (PBMCs) obtained from healthydonors.

In the following experiments, a full-length human Brachyury protein wasexpressed in insect cells via baculovirus expression and subsequentlypurified.

Dendritic cells (DCs) were prepared from PBMCs of healthy donors by5-day culture with GM-CSF and IL-4 and subsequently treated in vitrowith yeast-control (YVEC, see Example 4) or yeast-Brachyury (GI-6301,see Examples 1 and 2) (ratio yeast:DCs=1:1). After 48 hours, DCs wereharvested, irradiated (30 Gy) and used for stimulation of autologousPBMCs, at a ratio DC:PBMCs=1:10. On day 3, IL-2 (10 U/ml) was added tothe cultures. On day 7, stimulated T cells were harvested andsubsequently tested for IFN-γ production in response to autologous,irradiated PBMCs (ratio T cells:PBMCs=1:3) alone or in the presence of10 μg/ml of purified Brachyury protein or control human serum albuminprotein. Following 96 hours stimulation, supernatants were collected andevaluated for IFN-γ levels by ELISA assay. A total of 9 healthy donorswere evaluated, with 3/9 donors demonstrating Brachyury-specific CD4⁺T-cell responses post-stimulation in vitro with yeast-Brachyury-treatedDCs. Results for 3 positive cases are presented in Table 7 (valuesindicate the levels of IFN-γ in response to Brachyury protein, aftersubtracting background levels induced by stimulation with control HumanSerum Albumin protein; for donor 3, two cycles of stimulation wereperformed prior to evaluating response to Brachyury protein).

TABLE 7 ΔIFN-γ Donor ID DC stimulation (pg/ml) 1 Yeast-control 1500.0Yeast-Brachyury 2950.0 2 Yeast-control 13.4 Yeast-Brachyury 889.0 3Yeast-control 17.4 Yeast-Brachyury 102.8

Six additional healthy donors were evaluated for CD4⁺ T cell responsesto the Brachyury protein, following in vitro stimulation withyeast-Brachyury (GI-6301)-treated DCs by intracellular cytokine stainingof IFN-γ in CD4⁺ cells. Dendritic cells were prepared from PBMCs ofhealthy donors by 5-day culture with GMCSF and IL-4 and subsequentlytreated in vitro with yeast-control (YVEC) or yeast-Brachyury (GI-6301)(ratio yeast:DCs=1:1). After 48 hours, the DCs were harvested,irradiated (30 Gy) and used for stimulation of autologous PBMCs, at aratio DC:PBMCs=1:10. On day 3, IL-2 (10 U/ml) was added to the cultures.On day 7, stimulated T cells were harvested and subsequently tested forIFN-γ production in response to autologous PBMCs (ratio Tcells:PBMCs=1:3) alone or in the presence of 10 μg/ml of purifiedBrachyury protein or control human serum albumin protein. Following 2hours stimulation, BD GOLGISTOP™ Protein Transport Inhibitor (BDBiosciences) was added to the cultures. Following 4 hours stimulation,cells were harvested, permeabilized, and stained for CD4 and IFN-γutilizing anti-CD4 PerCP-Cy5.5 and anti-IFN-γ FITC antibodies (BDBiosciences). A total of 6 healthy donors were evaluated, with 2/6donors demonstrating Brachyury-specific CD4⁺ T-cell responsespost-stimulation in vitro with yeast-Brachyury treated DCs. Results forpositive cases are shown in Table 8 (values indicate the percentage of Tcells that were simultaneously positive for CD4 and intracellular IFN-γin response to control human serum albumin (HSA) or Brachyury protein,after subtracting background levels induced by stimulation with PBMCsalone).

TABLE 8 Number of % CD4⁺/IFN-γ⁺ cells Donor stimulations in vitro HSABrachyury 4 1 0.07 0.24 5 2 0.00 1.00

Example 10

The following example demonstrates that a yeast-Brachyury immunotherapycomposition expressing a Brachyury agonist antigen generatesBrachyury-specific T cells from a prostate cancer patient.

To generate a Brachyury-specific T-cell line, immature autologousdendritic cells (DCs) were exposed to the yeast-Brachyury immunotherapycomposition known at GI-6305 (see Example 3) at a ratio ofDCs:GI-6305=1:1 for 48 hours, and subsequently used as antigenpresenting cells (APCs) to stimulate autologous non-adherent cells at aneffector-to-APC ratio of 10:1. Cultures were incubated for 3 days at 37°C., in a humidified atmosphere containing 5% CO₂, and subsequentlysupplemented with recombinant human IL-2 at a concentration of 20 U/mlfor an additional 7 days. The 10-day culture constituted one in vitrostimulation (IVS) cycle. T cells were restimulated with GI-6305-exposedautologous DCs as described above on day 11, to begin the next IVScycle. GI-6305-exposed autologous DCs were used as APCs for three IVScycles. After the third IVS, irradiated (23,000 rads) autologousEBV-transformed B cells, pulsed with an agonist Brachyury peptide,WLLPGTSTV (SEQ ID NO:13), were used as APCs. A Brachyury-specific T cellline, denoted, T-2-BR-A, was established. This T cell line was used inthe immunoassays described below.

Table 9 demonstrates that Brachyury-specific T cells (T-2-BR-A) releasesignificant levels of IFN-γ after stimulation with allogeneic DCstreated with GI-6305, whereas control yeast (YVEC, see Example 4) didnot stimulate the release of IFN-γ by T-2-BR-A cells. Results areexpressed in pg/ml/10⁵ T cells. Briefly, allogeneic HLA-A2 positive DCsfrom a normal donor were treated with GI-6305 for 48 hours at variousyeast to DC ratios (indicated in Table 9) and then used to stimulateBrachyury agonist epitope-specific T cells (T-2-BR-A). In thisexperiment, the DC to T cell ratio was 1:10.

TABLE 9 Yeast/DC Dendritic Cells Treatment Ratio T Cells IFN-γ + Controlyeast  10:1 − <15.6 + Control yeast  10:1 + <15.6 − − − + 52.1 + GI-6305 10:1 − <15.6 + GI-6305  10:1 + 589.0 + GI-6305   5:1 − <15.6 + GI-6305  5:1 + 661.1 + GI-6305 2.5:1 − <15.6 + GI-6305 2.5:1 + 341.3 + GI-6305  1:1 − <15.6 + GI-6305   1:1 + 388.2

Table 10 demonstrates that Brachyury-specific T cells established byusing GI-6305 treated DCs can effectively lyse MDA-MB-231 breast cancercells that are HLA-A2 positive/Brachyury positive, but do not lyseASPC-1 pancreatic cancer cells that are HLA-A2 negative/Brachyurypositive. Briefly, the Brachyury-specific T cell line T-2-BR-A was usedat IVS-6 in an overnight cytotoxic T lymphocyte (CTL) assay againstMDA-MB-231 (HLA-A2⁺/Brachyury⁺) and ASPC-1 (HLA-A2⁻/Brachyury⁺) tumorcell targets, at the indicated effector:target (ET) ratios (see Table10). Results are expressed as the percentage of specific lysis.

TABLE 10 E:T ratio MDA-MB-231 ASPC-1   25:1 52.2 (2.8) −5.1 (2.6) 12.5:123.8 (1.4) 0.2 (5.6) 6.25:1 13.9 (4.4) 2.3 (3.3)

In another experiment, the ability of the T-2-BR-A cell line to bind toBrachyury-specific HLA-A2 tetramers was evaluated. Briefly, T-2-BR-Acells (used at IVS-4) were stained with a control tetramer or a tetramerspecific for the Brachyury agonist peptide. FIGS. 7A and 7B show that10.8% of CD8+ T cells in the T-2-BR-A cell line generated withGI-6305-treated DCs specifically bind to a Brachyury-HLA-A2 tetramer(FIG. 7B) and not to a control tetramer (FIG. 7A).

Perforin expression of the T-2-BR-A T cell line was analyzed by flowcytometry (perforin is a mediator of the cytolytic activity of cytotoxicT lymphocytes (CTLs)). Briefly, T cells were tested on day 5 afterrestimulation with Brachyury agonist peptide-pulsed autologous EBVtransformed B cells. FIG. 8 shows the expression of perforin in theT-2-BR-A cell line after stimulation with Brachyury agonistpeptide-pulsed autologous B cells, further demonstrating the cytotoxiccapability of this Brachyury-specific T cell line, which was generatedusing GI-6305-treated DCs.

Example 11

The following example describes a phase 1 clinical trial in subjectswith Brachyury-positive cancer.

An open-label, sequential dose-escalation, phase 1 clinical trial hasbeen initiated using the yeast-Brachyury immunotherapy composition knownas GI-6301, described in Examples 1, 2, and 4-9. Under this clinicaltrial protocol, 9-18 cancer patients (3-6 patients per dose cohort) areadministered the yeast-Brachyury immunotherapy composition known asGI-6301 in a sequential dose cohort escalation protocol utilizing doseranges of 4 Y.U. (1 Y.U.×4 sites, meaning that 1 Y.U. of GI-6301 isadministered at 4 different sites on the body of the patient eachvisit), 16 Y.U. (4 Y.U×4 sites) and 40 Y.U. (10 Y.U.×4 sites),administered subcutaneously. GI-6301 is administered at 2 week intervalsfor a total of 7 visits (˜3 months), and then monthly thereafter untilthe patients meet off-study criteria. An expansion cohort of patients(n=10) at maximum tolerated dose (MTD) or the observed best dose areselected for additional study. The results are monitoring safety andtolerability as a primary endpoint, and in the expanded cohort, whethera significant change in T cell precursors is detectable as measured byan increase in Brachyury-specific T cells in ELISpot assay andproliferation in response to Brachyury protein (e.g., Brachyury-specificCD8⁺ or CD4⁺ T cells emerging or expanding on treatment). As secondaryendpoints, clinical benefit, such as progression-free survival, clinicalradiographic response, reduction in serum markers, and/or reduction incirculating tumor cells is measured, as well as parameters of generalimmune activation, including frequency of immune cell subsets inperipheral blood (CD8⁺ memory/effector T cells, CD4⁺ memory/effector Tcells, Tregs, NK cells, DCs) and changes in serum levels of cytokines(e.g., IFN-γ, IL-10, IL-12, IL-2, IL-4, TGF-β, etc.).

GI-6301 is expected to be safe and well-tolerated with no significanttoxicities. In addition, GI-6301 is expected to producetreatment-emergent Brachyury-specific T cell responses or an improvementin pre-existing Brachyury-specific baseline T cell responses at leastsome or a majority of patients. Some patients are also expected to havestabilized disease.

In an additional study or an expansion of this study, theyeast-Brachyury immunotherapeutic composition known as GI-6305 (seeExample 3) is administered to an additional cohort of patients,utilizing the maximum tolerated dose or observed best dose determinedabove, and the same primary and secondary endpoints are measured.GI-6305 is also expected to be safe and well-tolerated with nosignificant toxicities, as well as produce treatment-emergentBrachyury-specific T cell responses or an improvement in pre-existingBrachyury-specific baseline T cell responses at least some or a majorityof patients. Some patients are also expected to have stabilized disease.

Example 12

The following example describes a phase 2 clinical trial usingyeast-Brachyury immunotherapeutic compositions.

A randomized phase 2 clinical trial in patients with breast cancer isrun using a yeast-Brachyury immunotherapeutic composition as describedin Example 1 and 2 (e.g., GI-6301) or in Example 3 (GI-6305). At least100 or more subjects with Stage I, II or III Brachyury-positive breastcancer are enrolled. Subject inclusion criteria can include subjectswith Grade 1, 2 or 3 cancers. Subject including criteria can alsoinclude subjects with “triple negative” breast cancer (cancers that arenegative for each of estrogen receptor (ER), progesterone receptor (PR)and HER2). Subject inclusion criteria can also include patients withlymph node-negative cancer.

The trial is run as a double-blind or open-label, placebo-controlled,multi-center trial. All patients receive standard of care therapy withtreatment arm patients receiving several serial injections ofyeast-Brachyury immunotherapeutic composition during treatment. Theprimary endpoint is recurrence free survival or overall survival.Additional endpoints can include antigen-specific T cell responses(e.g., Brachyury-specific CD8⁺ T cells emerging or expanding ontreatment), maintenance of lymph node negativity, progression tometastases, and Brachyury expression in tumor cells.

The yeast-Brachyury immunotherapeutic composition is expected to be safeand well-tolerated with no significant toxicities. In addition, theyeast-Brachyury immunotherapeutic composition is expected to producetreatment-emergent Brachyury-specific T cell responses and/or animprovement in pre-existing Brachyury-specific baseline T cell responsesin at least some or a majority of patients. Some or a majority ofpatients are also expected to have stabilized disease, maintain lymphnode negativity, and/or prevention, reduction or arrest in metastaticprogression.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing exemplary claims.

What is claimed is:
 1. A method to reduce tumor burden, increasesurvival, and/or inhibit tumor growth in an individual having a tumor,comprising administering to the individual an immunotherapeuticcomposition comprising: a) a whole, inactivated yeast; and b) aBrachyury fusion protein comprising the amino acid sequence of positions2-435 of SEQ ID NO:6 or the amino acid sequence of positions 2-435 ofSEQ ID NO:18; wherein the Brachyury fusion protein is expressed by theyeast; and wherein the composition elicits a Brachyury-specific T cellresponse.
 2. The method of claim 1, wherein the method further comprisessurgical resection of a tumor from the individual.
 3. The method ofclaim 1, wherein the tumor is of epithelial cell origin.
 4. The methodof claim 1, wherein the individual has a cancer selected from the groupconsisting of: breast cancer, small intestine cancer, stomach cancer,pancreatic cancer, kidney cancer, bladder cancer, uterine cancer,ovarian cancer, testicular cancer, lung cancer, colon cancer, prostatecancer, chronic lymphocytic leukemia (CLL), Epstein-Barr virustransformed B cells, Burkitt's lymphoma, Hodgkin's lymphoma, andmetastatic cancers thereof.
 5. The method of claim 1, wherein theindividual is being treated or has been treated with another therapy forcancer.
 6. The method of claim 1 further comprising administering one ormore additional immunotherapeutic compositions, wherein the each of theone or more additional immunotherapeutic compositions comprises anadditional cancer antigen.
 7. The method of claim 5, wherein the therapyis chemotherapy.
 8. The method of claim 5, wherein the therapy istargeted cancer therapy.
 9. The method of claim 5, wherein the therapyis radiation therapy.
 10. The method of claim 5, wherein the therapy isadoptive T cell transfer.
 11. The method of claim 5, wherein the therapyis administration of one or more additional immunotherapeuticcompositions.
 12. The method of claim 11, wherein the additionalimmunotherapeutic compositions comprise a yeast vehicle and a secondcancer antigen that does not include Brachyury antigen.
 13. The methodof claim 12, wherein the second cancer antigen is selected from thegroup consisting of: mutated Ras, carcinoembryonic antigen (CEA), MUC-1,EGFR, BCR-Abl, MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-2, PSMA,tyrosinase, TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA,HER-2/neu/c-erb/B2, hTERT, p73, B-RAF, adenomatous polyposis coli (APC),Myc, von Hippel-Lindau protein (VHL), Rb-1, Rb-2, androgen receptor(AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, pax3-fkhr, ews-fli-1, HERV-H,HERV-K, TWIST, Mesothelin, and NGEP.
 14. The method of claim 13, whereinthe second cancer antigen is selected from the group consisting of:mutated Ras, carcinoembryonic antigen (CEA), and MUC-1.
 15. The methodof claim 1 wherein the Brachyury fusion protein comprises the amino acidsequence of positions 2-435 of SEQ ID NO:6.
 16. The method of claim 1wherein the Brachyury fusion protein comprises the amino acid sequenceof positions 2-435 of SEQ ID NO:18.
 17. The method of claim 1 whereinthe Brachyury fusion protein comprises the amino acid sequence of SEQ IDNO:8.
 18. The method of claim 1 wherein the Brachyury fusion proteincomprises the amino acid sequence of SEQ ID NO:20.
 19. The method ofclaim 1, wherein the whole yeast is heat-inactivated.
 20. The method ofclaim 1, wherein the yeast is Saccharomyces cerevisiae.